Human IRAK-2

ABSTRACT

The present invention relates to a novel IRAK-2 protein which is a member of the IL-1 signaling pathway. In particular, isolated nucleic acid molecules are provided encoding the human IRAK-2 protein. IRAK-2 polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. Also provided are diagnostic methods for detecting IRAK-2 related disorders and therapeutic methods for treating IRAK-2 related disorders.

FIELD OF THE INVENTION

[0001] The present invention relates to a novel interleukin-1 receptorsignaling protein. More specifically, isolated nucleic acid moleculesare provided encoding a human interleukin-1 receptor associated kinase-2(IRAK-2). IRAK-2 polypeptides are also provided, as are vectors, hostcells and recombinant methods for producing the same.

BACKGROUND OF THE INVENTION

[0002] Interleukin-1 (IL-1).

[0003] Interleukin-1 (IL-1α and IL-1β) is a “multifunctional” cytokinethat affects nearly every cell type, and often in concert with othercytokines or small mediator molecules. (Dinarello, C. A., Blood87:2095-2147 (Mar. 15, 1996).) There are three members of the IL-1 genefamily: IL-1α, IL-1β, and IL-1 receptor antagonist (IL-1Rα). IL-1α andIL-1β are agonists and IL-1Ra is a specific receptor antagonist. IL-1αand β are synthesized as precursors without leader sequences. Themolecular weight of each precursor is 31 kD. Processing of IL-1α orIL-1β to “mature” forms of 17 kD requires specific cellular proteases.In contrast, IL-1Ra evolved with a signal peptide and is readilytransported out of the cells and termed secreted IL-1Ra (sIL-1Ra).

[0004] IL-1 Receptor and Ligands.

[0005] The receptors and ligands of the IL-1 pathway have been welldefined (for review, see Dinarello, C. A., FASEB J. 8:1314-1325 (1994);Sims, J. E. et al., Interleukin-1 signal transduction: Advances in Celland Molecular Biology of Membranes and Organelles, Vol. 3, JAI Press,Inc., Greenwich, Conn. (1994), pp. 197-222). Three ligands, IL-1α,IL-1β, and IL-1 receptor antagonist (IL-1Ra) bind three forms of IL-1receptor, an 80-kDa type I IL-1 receptor (IL-1RI) (Sims, J. E. et al.,Science 241:585-589 (1988)), a 68-kDa type II IL-1 receptor (IL-1RII)(McMahan, C. J. et al., EMBO J. 10:2821-2832 (1991)), and a soluble formof the type II IL-1R (sIL-1RII) (Colotta, F. et al., Science 261:472-475(1993)).

[0006] IL-1 Production in Various Disease States.

[0007] Increased IL-1 production has been reported in patients withvarious viral, bacterial, fungal, and parasitic infections;intravascular coagulation; high-dose IL-2 therapy; solid tumors;leukemias; Alzheimer's disease; HIV-1 infection; autoimmune disorders;trauma (surgery); hemodialysis; ischemic diseases (myocardialinfarction); noninfectious hepatitis; asthma; UV radiation; closed headinjury; pancreatitis; periodontitis; graft-versus-host disease;transplant rejection; and in healthy subjects after strenuous exercise.There is an association of increased IL-1β production in patients withAlzheimer's disease and a possible role for IL-1 in the release of theamyloid precursor protein (Vasilakos, J. P., et al., FEBS Lett. 354:289(1994)). However, in most conditions, IL-1 is not the only cytokineexhibiting increased production and hence the specificity of the IL-1findings as related to the pathogenesis of any particular disease islacking. In various disease states, IL-1β, but not IL-1α, is detected inthe circulation.

[0008] IL-1 in Therapy.

[0009] Although IL-1 has been found to exhibit many important biologicalactivities, it is also found to be toxic at doses that are close totherapeutic dosages (Dinarello, C. A., Blood87:2095-2147 (Mar. 15,1996)). In general, the acute toxicities of either isoform of IL-1 weregreater after intravenous compared with subcutaneous injection.Subcutaneous injection was associated with significant local pain,erythema, and swelling (Kitamura, T., & Takaku, F., Exp. Med. 7:170(1989); Laughlin, M. J., Ann. Hematol. 67:267 (1993)). Patientsreceiving intravenous IL-1 at doses of 100 ng/kg or greater experiencedsignificant hypotension. In patients receiving IL-1β from 4 to 32 ng/kgsubcutaneously, there was only one episode of hypotension at the highestdose level (Laughlin, M. J., Ann. Hematol. 67:267 (1993)).

[0010] Contrary to IL-1-associated myelostimulation in patients withnormal marrow reserves, patients with aplastic anemia treated with 5daily doses of IL-1α: (30 to 100 ng/kg) had no increases in peripheralblood counts or bone marrow cellularity (Walsh, C. E., et al., Br. J.Haematol 80:106 (1992)). IL-1 has been administered to patientsundergoing various regiments of chemotherapy to reduce the nadir ofneutropenia and thrombocytopenia.

[0011] Daily treatment with 40 ng/kg IL-1α from day 0 to day 13 ofautologous bone marrow or stem cells resulted in an earlier recovery ofneutropenia (median, 12 days; P<0.001) (Weisdorf, D., et al., Blood84:2044 (1994)). After 14 days of treatment, the bone marrow wassignificantly enriched with committed myeloid progenitor cells. Similarresults were reported in patients with AML receiving 50 ng/kg/d of IL-1βfor 5 days starting at the time of transplantation with purged ornonpurged bone marrow (Nemunaitis, J., et al., Blood 83:3473 (1994)).Injecting humans with low doses of either IL-1α or IL-1β confirms theimpressive pyrogenic and hypotension-inducing properties of themolecules.

[0012] IL-1 Signaling Mechanisms.

[0013] After binding to interleukin-1 (IL-1), the IL-1 receptor type I(IL-1RI) associates with the IL-1R Accessory Protein (IL-1RAcP) andinitiates a signaling cascade that results in the activation of NF-kB,(Greenfeder, S. A., et al., J. Biol. Chem. 270:13757-65 (1995); Sims, J.E., et al., Science 241:585-9 (1988); Korherr, C., et al., Eur. JImmunol. 27:262-7 (1997); Wesche, H., et al., J. Biol. Chem. 272:7727-31(1997); Freshney, N. W., et al., Cell 78:1039-49(1994); and Martin, M.,et al., Eur. J Immunol. 24:1566(1994)). Significant similarity existsbetween the IL-1R signaling pathway in mammals and the Toll signalingpathway in Drosophila. Toll, which shares sequence homology with thecytoplasmic domain of the IL-1RAcP, induces Dorsal activation (ahomologue of NF-kB) via the adapter protein Tube and the protein kinasePelle, (Galindo, R. L., et al, Development 121:2209-18 (1995); Norris,J. L. & Manley, J. L., Genes Devel. 10:862-72 (1996); Letsou, A., etal., EMBO 12:3449-3458 (1993); and Grosshans, J., et al., Nature372:563-566 (1994)); significantly the recently identified IRAK (IL-1RAssociated Kinase) is homologous to Pelle, (Cao, Z., et al., Science271:1128-31 (1996)). However, in mammalian cells, additional complexityis thought to exist based on the observation that multiple proteinkinase activities coprecipitate with the IL-1RI (Singh, R., et al., J.Clin. Invest. 100:419 (1997); and Eriksson, A., et al., Cytokine 7:649(1995)). Furthermore, given that in Drosophila the adapter protein Tubeinteracts with and regulates Pelle's activity, it is likely thatanalogous adapter/regulatory molecules might participate in IL-1signaling. There is a need in the art to characterize molecules involvedin the IL-1 signaling pathway.

[0014] Nuclear Factor Kappa B (NF-kB).

[0015] NF-kB is a member of a family of dimeric transcription factorsmade from monomers that have approximately 300 amino-acid Rel regionswhich bind to DNA, interact with each other, and bind the IkB inhibitors(for review, see Baeuerle and Baltimore, Cell 87:13-20 (1996)).Disregulation of NF-kB has been implicated in malignant transformationand hyperplasia (Gilmore et al., Oncogene 9:2391-2398 (1996)). NF-kBplays an important role in the antiviral response as a virus-inducibletranscriptional regulator of β-interferon, MHC class I, and inflammatorycytokine genes. NF-kB has also been shown to protect cells frompro-apoptotic stimuli (Beg et al., Nature 376:167-170 (1995)).

SUMMARY OF THE INVENTION

[0016] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the IRAK-2 polypeptide having theamino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4 or the aminoacid sequence encoded by the cDNA clone deposited in a bacterial host asATCC Deposit Number 209340 on Oct. 7, 1997.

[0017] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells and for using them forproduction of IRAK-2 polypeptides or peptides by recombinant techniques.

[0018] The invention further provides an isolated IRAK-2 polypeptidehaving an amino acid sequence encoded by a polynucleotide describedherein.

[0019] The present invention also provides a screening method foridentifying compounds capable of enhancing or inhibiting a cellularresponse induced by the IRAK-2, which involves contacting cells whichexpress the IRAK-2 with the candidate compound, assaying a cellularresponse, and comparing the cellular response to a standard cellularresponse, the standard being assayed when contact is made in absence ofthe candidate compound; whereby, an increased cellular response over thestandard indicates that the compound is an agonist and a decreasedcellular response over the standard indicates that the compound is anantagonist.

[0020] The invention provides a diagnostic method useful duringdiagnosis of a IRAK-2 or IL-1 disorder.

[0021] An additional aspect of the invention is related to a method fortreating an individual in need of an increased level of IRAK-2 activityin the body comprising administering to such an individual a compositioncomprising a therapeutically effective amount of an isolated IRAK-2polypeptide of the invention or an agonist thereof.

[0022] A still further aspect of the invention is related to a methodfor treating an individual in need of a decreased level of IRAK-2activity in the body comprising, administering to such an individual acomposition comprising a therapeutically effective amount of an IRAK-2antagonist.

BRIEF DESCRIPTION OF THE FIGURES

[0023]FIG. 1 shows the nucleotide (SEQ ID NO:1) and deduced amino acid(SEQ ID NO:2) sequences of IRAK-2α. The protein has a deduced molecularweight of about 65 kDa.

[0024]FIG. 2 shows the nucleotide (SEQ ID NO:3) and deduced amino acid(SEQ ID NO:4) sequences of IRAK-2β.

[0025]FIG. 3 shows the regions of similarity between the amino acidsequences of the IRAK-2α (SEQ ID NO:2) and IRAK-2β (SEQ ID NO:4)proteins and human IRAK (SEQ ID NO:5) and Pelle (SEQ ID NO:6). Alignmentwas performed with Custall software.

[0026]FIG. 4.

[0027]FIG. 4A shows that ectopic expression of IRAK-2 but not the mutantversion of IRAK-2 (1-96) activates NF-kB in 293 cells as measured byNF-kB reporter gene activity.

[0028]FIG. 4B shows that IRAK-2 (1-96) and IRAK-2 (97-590) inhibitIL-1Rs-induced NF-kB activity. Transfection with TRAF-2 (87-501) and NIK(KK429-430AA) expression vectors served as negative and positivecontrols, respectively. 0.1 μg of IL-1RI plus 0.1 μg of IL-1RAcP and 0.6μg of putative inhibitory expression constructs were transfected. Dataare expressed as percentage of relative IL-1Rs-induced NF-kB activity.

[0029]FIG. 5 shows that IRAK-2 induced NF-kB activity is specificallyabrogated by TRAF6 (289-522) but not TRAF2 (87-501). 293 cells weretransfected with 0.2 μg of IRAK-2 and increasing amounts of TRAFconstructs.

[0030]FIG. 6.

[0031]FIG. 6A shows that ectopic expression of MyD88 in 293 cellsresults in the induction of NF-kB activity. A mutant version of MyD88encoding a N-terminal region, MyD88 (1-152), was similarly capable ofinducing NF-kB activity albeit to a lesser extent; in contrast a mutantversion of MyD88 coding for amino acids 152 to the end, MyD88 (152-296)failed to induce any luciferase activity (not evident in graph). FIG. 6Bshows that MyD88-induced NF-kB activity was selectively inhibited by adominant negative version of TRAF6, TRAF6 (298-522) but not TRAF2(87-501). 0.1 μg of MyD88 and increasing amount of TRAF expressionconstructs were used. Data are expressed as percentage of relativeMyD88-induced NF-kB activity.

[0032] FIGS. 7A-B show that MyD88 (106-296) selectively inhibits IL-1Rs-but not TNFR2-induced NF-kB activity. TRAF6 (298-522) and the relatedTRAF2 (87-501) were used as controls. 0.5 μg receptors and increasingamounts of putative dominant negative expression constructs weretransfected. Data are expressed as percentage of relative IL-1Rs orTNFR2-induced NF-kB activity.

[0033] FIGS. 8A-C show that MyD88 dominant negative version, MyD88(152-296), abrogates IL-1Rs-induced but not IRAK-2-induced NF-kBactivity. Conversely IRAK-2 dominant negative versions, IRAK-2 (1-96)and IRAK-2 (97-590), significantly inhibit both IL-1Rs and MyD88-inducedNF-kB activity. 0.2 μg of inducer and 0.6 μg of dominant negativeexpression constructs were used in each transfection. Data are expressedas percentage of relative induced NF-kB activity.

[0034]FIG. 9 is a schematic representation of the molecular order ofmediators of the IL-1Rs-induced NF-kB activation.

[0035]FIG. 10 shows an analysis of the IRAK-2a amino acid sequence.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown. In the “Antigenic Index—Jameson-Wolf” graph,amino acid residues about 96 to about 193, about 207 to about 254, about293 to about 316, about 416 to about 472, and about 487 to about 541 inFIG. 1 (SEQ ID NO:2) correspond to the shown highly antigenic regions ofthe IRAK-2α protein.

[0036]FIG. 11 shows an analysis of the IRAK-2 β amino acid sequence.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown. In the “Antigenic Index—Jameson-Wolf” graph,amino acid residues 96 to about 193, about 207 to about 254, about 293to about 316, about 416 to about 472, about 487 to about 541, and about559 to about 619 in FIG. 2 (SEQ ID NO:4) correspond to the shown highlyantigenic regions of the IRAK-2β protein.

DETAILED DESCRIPTION

[0037] The present inventors have identified a human IRAK-2, IRAK-2α,and a splice variant thereof, IRAK-2β. Thus, the present inventionprovides isolated nucleic acid molecules comprising a polynucleotideencoding an IRAK-2 polypeptide having the amino acid sequence shown inSEQ ID NO:2. The present invention also provides isolated nucleic acidmolecules comprising a polynucleotide encoding an IRAK-2 polypeptidehaving the amino acid sequence shown in SEQ ID NO:4, which wasdetermined by sequencing a cloned cDNA. The IRAK-2α and IRAK-2β proteinsof the present invention shares sequence homology with IRAK (SEQ IDNO:5) and Pelle (SEQ ID NO:6). The nucleotide sequence shown in SEQ IDNO:3 was obtained by sequencing a cDNA clone, which was deposited onOct. 7, 1997 at the American Type Culture Collection, 12301 Park LawnDrive, Rockville, Md. 20852, and given accession number 209340. Thedeposited clone is inserted in the pBluescript SK(−) plasmid(Stratagene, LaJolla, Calif.) using the EcoRI and XhoI restrictionendonuclease cleavage sites.

[0038] Nucleic Acid Molecules

[0039] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequencer, and all amino acid sequences of polypeptides encoded byDNA molecules determined herein were predicted by translation of a DNAsequence determined as above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

[0040] Using the information provided herein, such as the nucleotidesequence in SEQ ID NO:1 or SEQ ID NO:3, a nucleic acid molecule of thepresent invention encoding an IRAK-2 polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the nucleic acid molecule described in SEQ ID NO:1 was discovered in acDNA library derived from HUVEC cells. The determined nucleotidesequence of the IRAK-2 cDNA of SEQ ID NO:1 contains an open readingframe encoding a protein of about 590 amino acid residues and a deducedmolecular weight of about 65 kDa. The nucleic acid molecule described inSEQ ID NO:3 was discovered in cDNA libraries derived from HUVEC cellsand activated neutrophils. The determined nucleotide sequence of theIRAK-2 cDNA of SEQ ID NO:3 contains an open reading frame encoding aprotein of about 625 amino acids. The IRAK-2 proteins shown in SEQ IDNO:2 and SEQ ID NO:4 are about 35-40 % identical and about 50-60 %similar to IRAK (SEQ ID NO:5).

[0041] As one of ordinary skill would appreciate, due to thepossibilities of sequencing errors, the predicted IRAK-2 polypeptideencoded by the deposited cDNA comprises about 625 amino acids, but maybe anywhere in the range of 600-650 amino acids.

[0042] As indicated, nucleic acid molecules of the present invention maybe in the form of RNA, such as mRNA, or in the form of DNA, including,for instance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

[0043] By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

[0044] Isolated nucleic acid molecules of the present invention includeDNA molecules comprising an open reading frame (ORF) shown in SEQ IDNO:1 or SEQ ID NO:3; and DNA molecules which comprise a sequencesubstantially different from those described above but which, due to thedegeneracy of the genetic code, still encode an IRAK-2 protein. Ofcourse, the genetic code is well known in the art. Thus, it would beroutine for one skilled in the art to generate such degenerate variants.

[0045] In addition, the present inventors have identified the followingcDNA clones related to extensive portions of SEQ ID NO:1 and SEQ IDNO:3: HPMCW18R (SEQ ID NO:7), HTADQ88R (SEQ ID NO:8), HNFEL57R (SEQ IDNO:9), HAPCM54R (SEQ ID NO:10), HNFFX36R (SEQ ID NO:11), HNFHL91R (SEQID NO:12), and HCE5L53R (SEQ ID NO:13).

[0046] The following public EST, which relates to portions of SEQ IDNO:1 and SEQ ID NO:3, has also been identified: Genbank Accession No.N52479, (SEQ ID NO:14).

[0047] In another aspect, the invention provides isolated nucleic acidmolecules encoding the IRAK-2 polypeptide having an amino acid sequenceas encoded by the cDNA clone contained in the plasmid deposited as ATCCDeposit No. 209340 on Oct. 7,1997. In a further embodiment, nucleic acidmolecules are provided encoding the full-length IRAK-2α or IRAK-2βpolypeptide lacking the N-terminal methionine. The invention alsoprovides an isolated nucleic acid molecule having the nucleotidesequence shown in SEQ ID NO:1 or SEQ ID NO:3 or the nucleotide sequenceof the IRAK-2 cDNA contained in the above-described deposited clone, ora nucleic acid molecule having a sequence complementary to one of theabove sequences. Such isolated molecules, particularly DNA molecules,are useful as probes for gene mapping, by in situ hybridization withchromosomes, and for detecting expression of the IRAK-2 gene in humantissue, for instance, by Northern blot analysis.

[0048] The present invention is further directed to fragments of theisolated nucleic acid molecules described herein. By a fragment of anisolated nucleic acid molecule having the nucleotide sequence of thedeposited cDNA or the nucleotide sequence shown in SEQ ID NO:1 or SEQ IDNO:3 is intended fragments at least about 15 nt, and more preferably atleast about 20 nt, still more preferably at least about 30 nt, and evenmore preferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250,1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, or 1700 nt in length arealso useful according to the present invention as are fragmentscorresponding to most, if not all, of the nucleotide sequence of thedeposited cDNA or as shown in SEQ ID NO:1 or SEQ ID NO:3. By a fragmentat least 20 nt in length, for example, is intended fragments whichinclude 20 or more contiguous bases from the nucleotide sequence of thedeposited cDNA or the nucleotide sequence as shown in SEQ ID NO:1 or SEQID NO:3.

[0049] Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding epitope-bearing portions of the IRAK-2protein. In particular, such nucleic acid fragments of the presentinvention include nucleic acid molecules encoding: a polypeptidecomprising amino acid residues from about 96 to about 193 in SEQ ID NO:2or SEQ ID NO:4; a polypeptide comprising amino acid residues from about207 to about 254 in SEQ ID NO:2 or SEQ ID NO:4; a polypeptide comprisingamino acid residues from about 293 to about 316 in SEQ ID NO:2 or SEQ IDNO: 4; a polypeptide comprising amino acid residues from about 416 toabout 472 in SEQ ID NO:2 or SEQ ID NO:4; a polypeptide comprising aminoacid residues from about 487 to about 541 in SEQ ID NO:2 or SEQ ID NO:4;and a polypeptide comprising amino acid residues from about 559 to about619 in SEQ ID NO:4. The inventors have determined that the abovepolypeptide fragments are antigenic regions of the IRAK-2 polypeptides.Methods for determining other such epitope-bearing portions of theIRAK-2 protein are described in detail below.

[0050] In another aspect, the invention provides an isolated nucleicacid molecule comprising a polynucleotide which hybridizes understringent hybridization conditions to a portion of the polynucleotide ina nucleic acid molecule of the invention described above, for instance,the cDNA clone contained in ATCC Deposit 209340. By “stringenthybridization conditions” is intended overnight incubation at 42° C. ina solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1× SSC at about 65° C.

[0051] By a polynucleotide which hybridizes to a “portion” of apolynucleotide is intended a polynucleotide (either DNA or RNA)hybridizing to at least about nucleotides (nt), and more preferably atleast about 20 nt, still more preferably at least about 30 nt, and evenmore preferably about 30-70 nt of the reference polynucleotide. Theseare useful as diagnostic probes and primers as discussed above and inmore detail below.

[0052] By a portion of a polynucleotide of “at least 20 nt in length,”for example, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:3).Of course, a polynucleotide which hybridizes only to a poly A sequence(such as the 3′ terminal poly(A) tract of the IRAK-2 cDNA shown in SEQID NO:1 or SEQ ID NO:3), or to a complementary stretch of T (or U)resides, would not be included in a polynucleotide of the invention usedto hybridize to a portion of a nucleic acid of the invention, since sucha polynucleotide would hybridize to any nucleic acid molecule containinga poly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

[0053] As indicated, nucleic acid molecules of the present inventionwhich encode an IRAK-2 polypeptide may include, but are not limited tothose encoding the amino acid sequence of the full-length polypeptide,by itself; the coding sequence for the full-length polypeptide andadditional sequences, such as those encoding a leader or secretorysequence, such as a pre-, or pro- or prepro- protein sequence; thecoding sequence of the full-length polypeptide, with or without theaforementioned additional coding sequences, together with additional,non-coding sequences, including for example, but not limited to intronsand non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals, forexample—ribosome binding and stability of mRNA; an additional codingsequence which codes for additional amino acids, such as those whichprovide additional functionalities. Thus, the sequence encoding thepolypeptide may be fused to a marker sequence, such as a sequenceencoding a peptide which facilitates purification of the fusedpolypeptide. In certain preferred embodiments of this aspect of theinvention, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (Qiagen, Inc.), among others,many of which are commercially available. As described in Gentz et al.,Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson et al., Cell 37:767-778(1984). As discussed below, other such fusion proteins include theIRAK-2 fused to Fc at the N- or C-terminus.

[0054] The present invention further relates to variants of the nucleicacid molecules of the present invention, which encode portions, analogsor derivatives of the IRAK-2 protein. Variants may occur naturally, suchas a natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

[0055] Such variants include those produced by nucleotide substitutions,deletions or additions, which may involve one or more nucleotides. Thevariants may be altered in coding regions, non-coding regions, or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions or additions.Especially preferred among these are silent substitutions, additions anddeletions, which do not alter the properties and activities of theIRAK-2 protein or portions thereof. Also especially preferred in thisregard are conservative substitutions.

[0056] Further embodiments of the invention include isolated nucleicacid molecules comprising a polynucleotide having a nucleotide sequenceat least 95% identical, and more preferably at least 96%, 97%, 98% or99% identical to (a) a nucleotide sequence encoding the polypeptidehaving the amino acid sequence in SEQ ID NO:2; (b) a nucleotide sequenceencoding the polypeptide having the amino acid sequence in SEQ ID NO:2,but lacking the N-terminal methionine; (c) a nucleotide sequenceencoding the polypeptide having the amino acid sequence in SEQ ID NO:4;(d) a nucleotide sequence encoding the polypeptide having the amino acidsequence in SEQ ID NO:4, but lacking the N-terminal methionine; (e) anucleotide sequence encoding the polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 209340;(f) a nucleotide sequence encoding the IRAK-2 polypeptide having theamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 209340, but lacking the N-terminal methionine; or (g) a nucleotidesequence complementary to any of the nucleotide sequences in (a), (b),(c), (d), (e), or (f).

[0057] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence encoding aIRAK-2 polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the IRAK-2polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

[0058] As a practical matter, whether any particular nucleic acidmolecule is at least 95%, 96%, 97%, 98% or 99% identical to, forinstance, the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3 orto the nucleotides sequence of the deposited cDNA clone can bedetermined conventionally using known computer programs such as theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). Bestfit uses the local homology algorithmof Smith and Waterman, Advances in Applied Mathematics 2: 482-489(1981), to find the best segment of homology between two sequences. Whenusing Bestfit or any other sequence alignment program to determinewhether a particular sequence is, for instance, 95% identical to areference sequence according to the present invention, the parametersare set, of course, such that the percentage of identity is calculatedover the full length of the reference nucleotide sequence and that gapsin homology of up to 5% of the total number of nucleotides in thereference sequence are allowed.

[0059] The present application is directed to nucleic acid molecules atleast 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in SEQ ID NO:1 or SEQ ID NO:3 or to the nucleic acid sequence ofthe deposited cDNA, irrespective of whether they encode a polypeptidehaving IRAK-2 activity. This is because even where a particular nucleicacid molecule does not encode a polypeptide having IRAK-2 activity, oneof skill in the art would still know how to use the nucleic acidmolecule, for instance, as a hybridization probe or a polymerase chainreaction (PCR) primer. Uses of the nucleic acid molecules of the presentinvention that do not encode a polypeptide having IRAK-2 activityinclude, inter alia, (1) isolating the IRAK-2 gene or allelic variantsthereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) tometaphase chromosomal spreads to provide precise chromosomal location ofthe IRAK-2 gene, as described in Verma et al., Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York (1988); and (3)Northern Blot analysis for detecting IRAK-2 mRNA expression in specifictissues.

[0060] Preferred, however, are nucleic acid molecules having sequencesat least 95%, 96%, 97%,98% or 99% identical to a nucleic acid sequenceshown in SEQ ID NO:1 or SEQ ID NO:3 or to a nucleic acid sequence of thedeposited cDNA which do, in fact, encode a polypeptide having IRAK-2protein activity. By “a polypeptide having IRAK-2 activity” is intendedpolypeptides exhibiting IRAK-2 activity in a particular biologicalassay. For example, IRAK-2 protein activity can be measured using theluciferase assay described in Cao et al., Nature 383: 443-446 (1996) andbelow in Example 1.

[0061] Briefly, cells which have been transfected with a nucleic acidencoding for a candidate polypeptide, such as human 293 cells, aretransfected with an ELAM-1-luciferase reporter plasmid. Luciferaseactivity is measured in these cells and compared to cells which havebeen transfected with the luciferase construct, but not with thecandidate polypeptide. A higher level of luciferase activity in cellswith the candidate polypeptide is indicative of IRAK-2 activity.

[0062] Of course, due to the degeneracy of the genetic code, one ofordinary skill in the art will immediately recognize that a large numberof the nucleic acid molecules having a sequence at least 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence of the deposited cDNAor a nucleic acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 willencode a polypeptide “having IRAK-2 protein activity.” In fact, sincedegenerate variants of these nucleotide sequences all encode the samepolypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving IRAK-2 protein activity. This is because the skilled artisan isfully aware of amino acid substitutions that are either less likely ornot likely to significantly effect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid).

[0063] For example, guidance concerning how to make phenotypicallysilent amino acid substitutions is provided in Bowie, J. U. et al.,“Deciphering the Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990), wherein the authorsindicate that proteins are surprisingly tolerant of amino acidsubstitutions.

[0064] Vectors and Host Cells

[0065] The present invention also relates to vectors which include theisolated DNA molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof IRAK-2 polypeptides or fragments thereof by recombinant techniques.

[0066] The polynucleotides may be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it maybe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

[0067] The DNA insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp andtac promoters, the SV40 early and late promoters and promoters ofretroviral LTRs, to name a few. Other suitable promoters will be knownto the skilled artisan. The expression constructs will further containsites for transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will preferablyinclude a translation initiating at the beginning and a terminationcodon (UAA, UGA or UAG) appropriately positioned at the end of thepolypeptide to be translated.

[0068] As indicated, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase or neomycin resistance for eukaryotic cell culture andtetracycline or ampicillin resistance genes for culturing in E. coli andother bacteria. Representative examples of appropriate hosts include,but are not limited to, bacterial cells, such as E. coli, Streptomycesand Salmonella typhimurium cells; fungal cells, such as yeast cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS and Bowes melanoma cells; and plant cells.Appropriate culture mediums and conditions for the above-described hostcells are known in the art.

[0069] Among vectors preferred for use in bacteria include pQE70, pQE60and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. Other suitable vectors will be readilyapparent to the skilled artisan.

[0070] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

[0071] The polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals, but alsoadditional heterologous functional regions. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Fcportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins, such as,hIL5-receptor has been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., Journal of Molecular Recognition, Vol. 8:52-58 (1995)and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270,No. 16:9459-9471 (1995).

[0072] The IRAK-2 protein can be recovered and purified from recombinantcell cultures by well-known methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography (“HPLC”) is employed for purification. Polypeptides ofthe present invention include naturally purified products, products ofchemical synthetic procedures, and products produced by recombinanttechniques from a prokaryotic or eukaryotic host, including, forexample, bacterial, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes.

[0073] IRAK-2 Polypeptides and Fragments

[0074] The invention further provides an isolated IRAK-2 polypeptidehaving the amino acid sequence encoded by the deposited cDNA, or theamino acid sequence in SEQ ID NO:2, or the amino acid sequence in SEQ IDNO:4, or a peptide or polypeptide comprising a portion of the abovepolypeptides.

[0075] It will be recognized in the art that some amino acid sequencesof the IRAK-2α or IRAK-2β polypeptides can be varied without significanteffect of the structure or function of the protein. If such differencesin sequence are contemplated, it should be remembered that there will becritical areas on the protein which determine activity.

[0076] Thus, the invention further includes variations of the IRAK-2α orIRAK-2β polypeptide which show substantial IRAK-2 polypeptide activityor which include regions of IRAK-2 protein such as the protein portionsdiscussed below. Such mutants include deletions, insertions, inversions,repeats, and type substitutions. As indicated above, guidance concerningwhich amino acid changes are likely to be phenotypically silent can befound in Bowie, J. U., et al., “Deciphering the Message in ProteinSequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310(1990).

[0077] Thus, the fragment, derivative or analog of the polypeptide ofSEQ ID NO:2 or SEQ ID NO:4, or that encoded by the deposited cDNA, maybe (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asan IgG Fc fusion region peptide or leader or secretory sequence or asequence which is employed for purification of the mature polypeptide ora proprotein sequence. Such fragments, derivatives and analogs aredeemed to be within the scope of those skilled in the art from theteachings herein.

[0078] Of particular interest are substitutions of charged amino acidswith another charged amino acid and with neutral or negatively chargedamino acids. The latter results in proteins with reduced positive chargeto improve the characteristics of the IRAK-2 protein. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)).

[0079] As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1). TABLE 1Conservative Amino Acid Substitutions. Aromatic Phenylalanine TryptophanTyrosine Hydrophobic Leucine Isoleucine Valine Polar GlutamineAsparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid GlutamicAcid Small Alanine Serine Threonine Methionine Glycine

[0080] Of course, the number of amino acid substitutions a skilledartisan would make depends on many factors, including those describedabove. Generally speaking, the number of amino acid substitutions forany given IRAK-2 polypeptide will not be more than 50, 40, 30, 20, 10, 5or 3.

[0081] Amino acids in the IRAK-2 proteins of the present invention thatare essential for function can be identified by methods known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as in vitro proliferative activity.

[0082] The polypeptides of the present invention are preferably providedin an isolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced and/orcontained within a recombinant host cell is considered isolated forpurposes of the present invention.

[0083] Also intended as an “isolated polypeptide” are polypeptides thathave been purified, partially or substantially, from a recombinant hostcell or a native source. For example, a recombinantly produced versionof the IRAK-2 polypeptide can be substantially purified by the one-stepmethod described in Smith and Johnson, Gene 67:31-40 (1988).

[0084] The polypeptides of the present invention include the apolypeptide comprising the polypeptide encoded by the deposited cDNA; apolypeptide comprising the polypeptide encoded by the deposited cDNA,but minus the N-terminal methionine; a polypeptide comprising aminoacids about 1 to about 590 in SEQ ID NO:2; a polypeptide comprisingamino acids about 2 to about 590 in SEQ ID NO:2; a polypeptidecomprising amino acids about 1 to about 625 in SEQ ID NO:4; apolypeptide comprising amino acids about 2 to about 625 in SEQ ID NO:4;as well as polypeptides which are at least 95% identical, and morepreferably at least 96%, 97%, 98% or 99% identical to those describedabove and also include portions of such polypeptides with at least 30amino acids and more preferably at least 50 amino acids.

[0085] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a reference amino acid sequence of a IRAK-2polypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the IRAK-2 polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0086] As a practical matter, whether any particular polypeptide is atleast 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in SEQ ID NO:2 or SEQ ID NO:4 or to the amino acidsequence encoded by deposited cDNA clone can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5 % of the total number of amino acidresidues in the reference sequence are allowed.

[0087] The polypeptide of the present invention are useful as amolecular weight marker on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart.

[0088] In another aspect, the invention provides a peptide orpolypeptide comprising an epitope-bearing portion of a polypeptide ofthe invention. The epitope of this polypeptide portion is an immunogenicor antigenic epitope of a polypeptide described herein. An “immunogenicepitope” is defined as a part of a protein that elicits an antibodyresponse when the whole protein is the immunogen. On the other hand, aregion of a protein molecule to which an antibody can bind is defined asan “antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).

[0089] As to the selection of peptides or polypeptides bearing anantigenic epitope (i.e., that contain a region of a protein molecule towhich an antibody can bind), it is well known in that art thatrelatively short synthetic peptides that mimic part of a proteinsequence are routinely capable of eliciting an antiserum that reactswith the partially mimicked protein. See, for instance, Sutcliffe, J.G., Shinnick, T. M., Green, N. and Learner, R. A. (1983) Antibodies thatreact with predetermined sites on proteins. Science 219:660-666.Peptides capable of eliciting protein-reactive sera are frequentlyrepresented in the primary sequence of a protein, can be characterizedby a set of simple chemical rules, and are confined neither toimmunodominant regions of intact proteins (i.e., immunogenic epitopes)nor to the amino or carboxyl terminals.

[0090] Antigenic epitope-bearing peptides and polypeptides of theinvention are therefore useful to raise antibodies, including monoclonalantibodies, that bind specifically to a polypeptide of the invention.See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777.

[0091] Antigenic epitope-bearing peptides and polypeptides of theinvention preferably contain a sequence of at least seven, morepreferably at least nine and most preferably between about at leastabout 15 to about 30 amino acids contained within the amino acidsequence of a polypeptide of the invention.

[0092] Non-limiting examples of antigenic polypeptides or peptides thatcan be used to generate IRAK-2-specific antibodies include: apolypeptide comprising amino acid residues from about 96 to about 193 inSEQ ID NO:2 or SEQ ID NO:4; a polypeptide comprising amino acid residuesfrom about 207 to about 254 in SEQ ID NO:2 OR SEQ ID NO:4; a polypeptidecomprising amino acid residues from about 293 to about 316 in SEQ IDNO:2 or SEQ ID NO: 4; a polypeptide comprising amino acid residues fromabout 416 to about 472 in SEQ ID NO:2 or SEQ ID NO:4; a polypeptidecomprising amino acid residues from about 487 to about 541 in SEQ IDNO:2 or SEQ ID NO:4; and a polypeptide comprising amino acid residuesfrom about 559 to about 619 in SEQ ID NO:4. As indicated above, theinventors have determined that the above polypeptide fragments areantigenic regions of the IRAK-2 protein.

[0093] The epitope-bearing peptides and polypeptides of the inventionmay be produced by any conventional means. Houghten, R. A. (1985)General method for the rapid solid-phase synthesis of large numbers ofpeptides: specificity of antigen-antibody interaction at the level ofindividual amino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135. This“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

[0094] As one of skill in the art will appreciate, IRAK-2 polypeptidesof the present invention and the epitope-bearing fragments thereofdescribed above can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric IRAK-2 protein orprotein fragment alone (Fountoulakis et al., J. Biochem 270:3958-3964(1995)).

[0095] Screening Assays

[0096] The present inventors have shown that IRAK-2 mediates NF-kBactivation induced by IL-1R stimulation. NF-kB is an ubiquitoustranscription factor which has been shown to activate transcription ofenzymes, such as cyclooxygenase-2 (Newton et al., Biochem. Biophys. Res.Commun. 237(1):28-32 (1997)); cytokines, such as RANTES (Moriuchi etal., J. Immunol. 158(7):3483-3491 (1997)); adhesion molecules, such asE-selectin (ELAM-1) (Read et al., J. Biol. Chem. 272(5):2753-2761(1997)); and other molecules. The normal functions of NF-kB includecommunication between cells, embryonal development, the response tostress, inflammation and viral infection, and the maintenance of celltype specific expression of genes (for review, see Wulczyn et al., J.Mol. Med. 74(12):749-769 (1996)). Upregulation of NF-kB could be used totreat viral infections, such as HIV ((Moriuchi et al., J. Immunol.158(7):3483-3491 (1997)), and damage caused by oxidative stress (Renardet al., Biochem. Pharmacol. 53:149-160 (1997)). Disregulation of NF-kBactivation has been linked to adult respiratory distress syndrome,sepsis syndrome, asthma, rheumatoid arthritis, inflammatory boweldisease, malignant transformation and hyperplasia (Blackwell et al., Am.J. Respir. Cell. Mol. Biol. 17(1):3-9 (1997); Barnes, Int. J. Biochem.Cell. Biol. 29(6):867-870 (1997); and Gilmore et al., Oncogene9:2391-2398 (1996)). Accordingly, inhibitors of NF-kB could be used totreat these disorders. Several inhibitors of NF-kB have been identified,including antioxidants such as alpha-tocopherol (Erl et al., Am. J.Physiol. 273:H634-H640 (1997)), and glucocorticoids, such asdexamethasone (Wang et al., J. Immunol. 159:534-537 (1997))).

[0097] Thus, the present invention also provides a screening method fordetermining whether a compound of interest is an agonist or antagonistof the IRAK-2 pathway. This method involves contacting cells whichexpress IRAK-2, either exogenously or endogenously, with a compound ofinterest, assaying NF-kB mediated transcription, and comparing the NF-kBmediated transcription to a standard response. The standard response isthe level of NF-kB mediated transcription in cells expressing IRAK-2that have not been contacted with the compound of interest, whereby anincrease in NF-kB mediated transcription over the standard indicatesthat the compound of interest is an agonist of the IRAK-2 pathway and adecrease in NF-kB mediated transcription under the standard indicatesthat the compound of interest is an antagonist of the IRAK-2 pathway.

[0098] By “assaying NF-kB mediated transcription” is intendedqualitatively or quantitatively measuring NF-kB mediated transcription.By the invention, the compound of interest is an agonist of the IRAK-2pathway if NF-kB mediated transcription is enhanced over that observeddue to IRAK-2 in the absence of the compound of interest and thecompound of interest is an antagonist of the IRAK-2 pathway if NF-kBmediated transcription is diminished compared to that observed due toIRAK-2 in the absence of the compound of interest. Since IRAK-2activates NF-kB transcription, any in vitro or in vivo assay whichmeasures NF-kB activity can be used in this method.

[0099] For example, a construct encoding for IRAK-2 is transfected intoa cell, along with a construct containing a reporter gene which is underthe control of a promoter which is activated in the presence of NF-kB.Any reporter gene which is known in the art can be used in this assay.Examples of reporter genes useful in this assay include, but are notlimited to, luciferase, P-galactosidase, and chloramphenicolacetyltransferase. NF-kB-responsive promoters can include one or morebinding sites for NF-kB. Examples of promoters which are sensitive toNF-kB include, but are not limited to, the promoter for ELAM-1 and thepromoter for RANTES. After transfection of the constructs, the cell iscontacted with a compound of interest, and the reporter gene expressionis measured and compared to the reporter gene expression seen in cellswhich have not been contacted with the compound of interest. An increasein reporter gene expression in cells which have been contacted with thecompound of interest indicates that the compound is an agonist of theIRAK-2 pathway. A decrease in reporter gene expression in cells whichhave been contacted with the compound of interest indicates that thecompound is an antagonist of the IRAK-2 pathway.

[0100] IRAK-2 Related Disorder Diagnosis

[0101] For IRAK-2 related disorders, it is believed that substantiallyaltered (increased or decreased) levels of IRAK-2 gene expression can bedetected in tissues taken from a mammal having such a disorder, relativeto a “standard” mammal, i.e., a mammal of the same species not havingthe disorder. Thus, the invention provides a diagnostic method usefulduring diagnosis of an IRAK-2 related disorder, which involves assayingthe expression level of the gene encoding the IRAK-2 protein inmammalian cells or body fluid and comparing the gene expression levelwith a standard IRAK-2 gene expression level, whereby an increase in thegene expression level over the standard is indicative of certaindisorders.

[0102] IRAK-2 related disorders are believe to include, but are notlimited to, leukemia, lymphoma, rheumatoid arthritis, sarcoidosis,tuberculosis, onchocerciasis, allergies, various bacterial infections,arteriosclerosis, autoimmune diseases, and inflammatory diseases.

[0103] Where a diagnosis has already been made according to conventionalmethods, the present invention is useful as a prognostic indicator,whereby patients exhibiting enhanced IRAK-2 gene expression willexperience a worse clinical outcome relative to patients expressing thegene at a lower level.

[0104] By “assaying the expression level of the gene encoding the IRAK-2protein” is intended qualitatively or quantitatively measuring orestimating the level of the IRAK-2 protein or the level of the mRNAencoding the IRAK-2 protein in a first biological sample either directly(e.g., by determining or estimating absolute protein level or mRNAlevel) or relatively (e.g., by comparing to the IRAK-2 protein level ormRNA level in a second biological sample).

[0105] Preferably, the IRAK-2 protein level or mRNA level in the firstbiological sample is measured or estimated and compared to a standardIRAK-2 protein level or mRNA level, the standard being taken from asecond biological sample obtained from an individual not having thedisorder. As will be appreciated in the art, once a standard IRAK-2protein level or mRNA level is known, it can be used repeatedly as astandard for comparison.

[0106] By “biological sample” is intended any biological sample obtainedfrom an individual, cell line, tissue culture, or other source whichcontains IRAK-2 protein or mRNA. Biological samples include mammalianbody fluids (such as sera, plasma, urine, synovial fluid and spinalfluid) which contain IRAK-2 protein, and ovarian, prostate, heart,placenta, pancreas liver, spleen, lung, breast and umbilical tissue.

[0107] Preferred mammals include monkeys, apes, cats, dogs, cows, pigs,horses, rabbits and humans. Particularly preferred are humans.

[0108] Total cellular RNA can be isolated from a biological sample usingthe single-step guanidinium-thiocyanate-phenol-chloroform methoddescribed in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987).Levels of mRNA encoding the IRAK-2 protein are then assayed using anyappropriate method. These include Northern blot analysis (Harada et al.,Cell 63:303-312 (1990)), S1 nuclease mapping (Fujita et al., Cell49:357-367 (1987)), the polymerase chain reaction (PCR), reversetranscription in combination with the polymerase chain reaction (RT-PCR)(Makino et al., Technique 2:295-301 (1990)), and reverse transcriptionin combination with the ligase chain reaction (RT-LCR).

[0109] Assaying IRAK-2 protein levels in a biological sample can occurusing antibody-based techniques. For example, IRAK-2 protein expressionin tissues can be studied with classical immunohistological methods(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,et al., J. Cell. Biol. 105:3087-3096 (1987)).

[0110] Other antibody-based methods useful for detecting IRAK-2 proteingene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA).

[0111] Suitable labels are known in the art and include enzyme labels,such as, Glucose oxidase, and radioisotopes, such as iodine (¹²⁵I,¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), andtechnetium (^(99m)Tc), and fluorescent labels, such as fluorescein andrhodamine, and biotin.

[0112] Modes of Administration

[0113] It will be appreciated that conditions caused by a decrease inthe standard or normal level of IRAK-2 activity in an individual can betreated by administration of IRAK-2 protein. Thus, the invention furtherprovides a method of treating an individual in need of an increasedlevel of IRAK-2 activity comprising administering to such an individuala pharmaceutical composition comprising an effective amount of anisolated IRAK-2 polypeptide of the invention effective to increase theIRAK-2 activity level in such an individual.

[0114] As a general proposition, the total pharmaceutically effectiveamount of IRAK-2 polypeptide administered parenterally per dose will bein the range of about 1 μg/kg/day to 10 mg/kg/day of patient bodyweight, although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the IRAK-2 polypeptide is typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed.

[0115] Pharmaceutical compositions containing the IRAK-2 of theinvention may be administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, drops or transdermal patch), bucally, or as an oralor nasal spray. By “pharmaceutically acceptable carrier” is meant anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

[0116] Chromosome Assays

[0117] The nucleic acid molecules of the present invention are alsovaluable for chromosome identification. The sequence is specificallytargeted to and can hybridize with a particular location on anindividual human chromosome. The mapping of DNAs to chromosomesaccording to the present invention is an important first step incorrelating those sequences with genes associated with disease.

[0118] In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a IRAK-2 protein gene. Thiscan be accomplished using a variety of well known techniques andlibraries, which generally are available commercially. The genomic DNAthen is used for in situ chromosome mapping using well known techniquesfor this purpose.

[0119] In addition, in some cases, sequences can be mapped tochromosomes by preparing PCR primers (preferably 15-25 bp) from thecDNA. Computer analysis of the 3′ untranslated region of the gene isused to rapidly select primers that do not span more than one exon inthe genomic DNA, thus complicating the amplification process. Theseprimers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes.

[0120] Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom the cDNA as short as 50 or 60 bp. For a review of this technique,see Verma et al., Human Chromosomes: A Manual Of Basic Techniques,Pergamon Press, New York (1988).

[0121] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance In Man, available on-line throughJohns Hopkins University, Welch Medical Library. The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0122] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0123] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLES Example 1 Characterization of IRAK-2α

[0124] A novel partial human cDNA was identified that showed significanthomology to both IRAK and Pelle. Screening of a human HUVEC cDNA libraryresulted in the isolation of a full length cDNA clone; analysis of thenucleotide sequence revealed an open reading frame encoding a 590 aminoacids (aa) protein with a calculated MW of 65 kDa (FIG. 1). Clustallalignment analysis showed significant homology to both IRAK and Pelle(FIG. 3). Given its sequence and functional similarity to IRAK themolecule was designated IRAK-2. Northern blot analysis revealed a singleIRAK-2 transcript expressed in a variety of tissues whose size (about4Kbp) was consistent with that of the cDNA.

[0125] Ectopic expression of IRAK-2 in human 293 cells induced NF-kBactivation as determined by relative luciferase activity of a NF-kBresponsive construct. Truncated versions of IRAK-2 encoding amino acidresidues 1 to 96 of SEQ ID NO:2 [IRAK-2 (1-96)] or amino acid residues97 to 590 of SEQ ID NO:2 [IRAK-2 (97-590)] failed to induce anyluciferase activity suggesting that integrity of the molecule wasessential for its function (FIG. 4A). Deletional analysis has previouslyshown that a mutant version of Pelle analogous to IRAK-2 (97-590) isalso inactive leading to the suggestion that Pelle's recruitment to theplasma membrane through its N-terminal domain is necessary for itssubsequent function (Galindo, R. L., et al., Development 121:2209-2218(1995)). Given this, it was tested whether IRAK-2 (1-96) or IRAK-2(97-590) could act as dominant negative inhibitors of IL-1R-inducedNF-kB activity. Coexpression of IL-1RI and IL-1RAcP (IL-1Rs for clarity)strongly induced NF-kB activity. Surprisingly, both IRAK-2 (1-96) andIRAK-2 (97-590) inhibited IL-1Rs-induced NF-kB activity. A dominantnegative mutant version of the downstream kinase NIK that is implicatedin IL-1R-induced NF-kB activation was used as a positive control; theunrelated adapter molecule TRAF2 (298-522) was used as a negativecontrol (FIG. 4B).

[0126] Given the sequence similarity shared by IRAK and IRAK-2, and thefunctional involvement of IRAK-2 in IL-1Rs-induced NF-kB activity, itwas analyzed whether IRAK-2 was recruited to the IL-1R signalingcomplex. Interestingly, while IRAK preferentially coprecipitated withIL-1 RAcP, IRAK-2 preferentially bound to the IL-1RI. In contrast, amutant version of IRAK-2 lacking the first 96 amino acid residues[IRAK-2 (97-590)] failed to associate with IL-1RI suggesting that itsN-terminal domain docks with the cytoplasmic domain of IL-1RI.Confirming this was the finding that a truncated form of IRAK-2 codingfor the first 96 amino acid residues [IRAK-2 (1-96)] specificallycoprecipitated with IL-1RI.

[0127] Certain members of the TRAF adapter family mediate NF-kBactivation induced by a number of cytokine receptors. TRAF2, for exampleplays a critical role in TNFR1 and -2 mediated NF-kB activation. TRAF6has recently been implicated in the IL-1 signaling pathway and shown tocomplex with IRAK (Cao, Z., et al., Nature 383:443-6 (1996)). It wastherefore determined if IRAK-2 interacted with TRAF6 when coexpressed in293T cells. Both IRAK and IRAK-2 coprecipitated with TRAF6 but not withthe related TRAF2. A dominant negative version of TRAF6 [TRAF6(298-522)] which inhibits IL-1-induced NF-kB activity, also bound bothIRAK and IRAK-2. Further, IRAK-2-induced NF-kB activity was specificallyinhibited by dominant negative TRAF6 (298-522) but not by a dominantnegative version of TRAF2 [TRAF2 (87-501)] (FIG. 5). These data are inkeeping with TRAF6 acting downstream of IRAK-2, in the IL-1 mediatedNF-kB signaling pathway.

[0128] Additional putative proximally participating adapters/regulatorswere sought by systematically looking for proteins showing homology toeither Tube or IL-1RAcP. BLAST searches of the public data base revealedthe cytoplasmic domain of the IL-1RAcP to possess significant homologyto MyD88 (Lord, K., et al., Oncogene 5:1095 (1990)). Sequence similaritybetween MyD88, IL-1RI and Toll has previously been reported, but thefunctional significance of this homology has been obscure.Interestingly, the MyD88 polypeptide has a modular structure composed oftwo fused module types: a N-terminal “interaction domain” (or DD forDeath Domain that was initially defined in proteins involved inprogrammed cell death), (Feinstein, E., et al., Trends Biochem. Sci.20:342-4 (1995); and Hofmann, K. & Tschopp, J., et al., FEBS Letters371:321 (1995)) and a C-terminal domain related to the cytoplasmicregion of IL-1 RAcP, IL-1RI, Toll and the recently identified human Tollhomologue (Hardiman, G., et al., Oncogene 13:2467-75 (1996); Hultmark,D., Biochem. Biophys. Res. Commun. 199:144 (1994); Bonnert, T., et al.,FEBS lett. 402:81-84 (1997); and Medzhitov, R., et al., Nature 388:394(1997)). Given the presence of these two distinct domains it washypothesized that MyD88 might simultaneously connect a transmembranereceptor belonging to the IL-1R family with a downstream signalingmediator. To test this, the role of human MyD88 was functionallycharacterized.

[0129] Ectopic expression of MyD88 in 293 cells strongly induced NF-kBactivity in a dose dependent manner. Similarly, a truncated version ofMyD88 encoding the N-terminal domain (DD), MyD88 (1-151), activatedNF-kB albeit to a lesser extent. In contrast, the C-terminal region,MyD88 (152-296) did not induce any luciferase activity (FIG. 6A).Significantly, MyD88-induced NF-kB activity was specifically inhibitedby TRAF6 but not TRAF2 dominant negative expression constructssuggesting that TRAF6 and MyD88 likely participate in the same signalingpathway and that TRAF6 functions downstream of MyD88 (FIG. 6B). It wasnext tested whether MyD88 (152-296) could act as a dominant negativeinhibitor of IL-1Rs-induced NF-kB activity; MyD88 (152-296) specificallyinhibited IL-1Rs-induced but not TNFR2-induced NF-kB activation. Adominant negative version of TRAF6 [TRAF6 (289-522)] similarly inhibitedIL-1Rs-induced but not TNFR2-induced NF-kB activation; in contrast, adominant negative version of TRAF2 [TRAF2 (87-501)] abrogatedTNFR2-induced, but not IL-1Rs-induced, NF-kB activity confirming thespecificity of effects observed with MyD88 (152-296).

[0130] Given the significant sequence homology existing between MyD88and the IL-1RAcP, it was investigated whether the two could interact.Upon coexpression in 293T cells, MyD88 and IL-1RAcP formed animmunoprecipitable complex. IL-1RI, which shows weaker sequencesimilarity to MyD88, did not associate with MyD88 under theseexperimental conditions. Domain mapping studies revealed that thesequence homologous C-terminal region of MyD88 was sufficient forbinding to the IL-1RAcP cytoplasmic domain (FIGS. 7A-7B) consistent witha hemophilic interaction.

[0131] In an effort to molecularly order the proximal components of theIL-1R signaling complex identified herein, it was tested whether thedominant negative mutant versions of MyD88 and IRAK-2 could inhibit theactive forms of the others. A dominant negative version of MyD88completely abrogated IL-1Rs-induced NF-kB activation but failed toinhibit IRAK-2-induced NF-kB activation (FIGS. 8A-8C). On the otherhand, dominant negative versions of IRAK-2, significantly inhibited bothIL-1Rs- and MyD88-induced NF-kB activity. These results are consistentwith MyD88 acting upstream of IRAK-2 in the IL-1R signaling pathway.

[0132] Given the presence of a N-terminal “interaction domain” (DD) inboth MyD88 and IRAK-2 (Feinstein, E., et al., supra; and Hofmann, K. &Tschopp, J., supra)) it was tested whether these two proteins couldinteract. It was found that MyD88 specifically coprecipitated withIRAK-2. Significantly a truncated version of IRAK-2 lacking theN-terminal domain (DD) [IRAK-2 (97-590)], that failed to induce NF-kBactivation, also failed to associate with MyD88; similarly, the versionof MyD88 (152-296) that was unable to induce NF-kB activity, was alsoimpaired in its ability to bind IRAK-2 lending functional credence tothis interaction.

[0133] Taken together these results support a model wherein MyD88 actsas an adapter/regulator in the IL-1R signaling complex by independentlyinteracting with IL-1RAcP and IRAK-2. However, we were unable, underthese experimental conditions, to assemble a multimolecular complexbetween MyD88, IRAK-2 and the IL-1Rs. This is consistent with thepossibility that MyD88 is only transiently recruited to the IL-1Rsignaling complex where it subsequently regulates IRAK-2's activity.

[0134] Methods

[0135] cDNA Cloning and Analysis.

[0136] A partial cDNA clone was used to screen a human HUVEC cDNAlibrary. Hybridizing clones were characterized by automated DNAsequencing. Alternatively the sequence corresponding to aa, 391 to 570of IL-1 RAcP was used to search the NCBI Gene Bank nr database. Humanand murine MyD88 cDNAs were identified as having statisticallysignificant homology to IL-1RAcP. Sequence assembly, comparison andalignment were performed using DNASTAR software.

[0137] Expression Vectors.

[0138] Mammalian expression vectors encoding Flag-TRAF6, Flag-TRAF6(289-522), Flag-TRAF2, Flag-TRAF2 (87-501), NIK (KK429-430AA),ELAM-Luciferase reporter plasmid, Flag-IL-1RAcP and IRAK have beenpreviously described ((Cao, Z., et al., Nature 383:443-6 (1996);Chinnaiyan, A., et al., Science 274:990-92 (1996); Malinin, N. L., etal., Nature 385:5:540-4 (1997); and Rothe, M., et al., Science269:1424-7 (1995)). AU1-IRAK-2 (1-96), AU1-MyDS88, AU-1-MyD88 (152-296)and HA-MyD88 (1-151) were PCR amplified from a HUVEC cDNA library usingcustom-made oligonucleotide primers encoding the AU1 or HA epitope tag.Amplified fragments were cloned into the mammalian expression vectorpCDNA3 (Invitrogen). IRAK-2-MyC and IRAK-2 (97-590)-MyC were obtained hyPCR amplification and cloned in frame into pCDNA3-MyC-His vector(Invitrogen). Flag-IL-1RI and Flag-ΔIL-1RI were similarly obtained byPCR amplification from the HUVEC cDNA library and sub cloned in frameinto pCMV-1-Flag expression vector.

[0139] Transfection and Coimmunoprecipitation.

[0140] Human embryonic 293 or 293T cells were transiently transfected bycalcium phosphate method with the indicated plasmids. The total amountof DNA was kept constant. 24-36 hours after transfection, cells werelysed in 0.5 ml buffer (1% NP40, 150 mM NaCl, 50 mM Tris, 1 mM EDTA andprotease inhibitors cocktail). Cell lysates were adjusted to 0.7 M NaCland the indicated antibodies were added for 1 to 4 hours. Immunecomplexes were precipitated by the addition of protein-G-Sepharose(Sigma). After extensive washing, the Sepharose heads were boiled insample buffer and the eluted proteins fractionated by SDS-PAGE.Subsequent protein immunoblotting was performed as described(Chinnaiyan, A., et al., Cell 81:505-12 (1995)).

[0141] NF-kB Luciferase Assay.

[0142] Cells were transfected with 0.1 μg ELAM-Luciferase reporterplasmid, 0.2 μg pCMV-βGal and the indicated expression vectors; totalamount of transfected DNA was kept constant by supplementation withempty vector. Relative NF-kB activity was calculated by normalizingrelative luciferase activity with βGal activity as previously described(Cao, Z. et al., Nature 383:443-446 (1996).

Example 2 Tissue Distribution of IRAK-2 mRNA Expression

[0143] Northern blot analysis is carried out to examine IRAK-2 geneexpression in human tissues, using methods described by, among others,Sambrook et al., cited above. A cDNA probe containing the nucleotidesequence corresponding to the open reading frame of the IRAK-2a protein(SEQ ID NO:1) is labeled with ³²P using the rediprime™ DNA labelingsystem (Amersham Life Science), according to manufacturer'sinstructions. After labeling, the probe is purified using a CHROMASPIN-100™ column (Clontech Laboratories, Inc.), according tomanufacturer's protocol number PT1200-1. The purified labeled probe isthen used to examine various human tissues for IRAK-2 mRNA.

[0144] Multiple Tissue Northern (MTN) blots containing various humantissues (H) are obtained from Clontech and examined with the labeledprobe using ExpressHyb™ hybridization solution (Clontech) according tomanufacturer's protocol number PT1190-1. Following hybridization andwashing, the blots are mounted and exposed to film at −70° C. overnight,and films developed according to standard procedures.

[0145] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples.

[0146] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[0147] The entire disclosure of all publications (including patents,patent applications, journal articles, laboratory manuals, books, orother documents) cited herein are hereby incorporated by reference.

1 14 1806 base pairs nucleic acid double linear DNA (genomic) CDS34..1803 1 GCAGGCGCGC CGGAGCCGGC CCCGTAGCGT GCC ATG GCC TGC TAC ATC TACCAG 54 Met Ala Cys Tyr Ile Tyr Gln 1 5 CTG CCC TCC TGG GTG CTG GAC GACCTG TGC CGC AAC ATG GAC GCG CTC 102 Leu Pro Ser Trp Val Leu Asp Asp LeuCys Arg Asn Met Asp Ala Leu 10 15 20 AGC GAG TGG GAC TGG ATG GAG TTC GCCTCC TAC GTG ATC ACA GAC CTG 150 Ser Glu Trp Asp Trp Met Glu Phe Ala SerTyr Val Ile Thr Asp Leu 25 30 35 ACC CAG CTG CGG AAG ATC AAG TCC ATG GAGCGG GTG CAG GGT GTG AGC 198 Thr Gln Leu Arg Lys Ile Lys Ser Met Glu ArgVal Gln Gly Val Ser 40 45 50 55 ATC ACG CGG GAG CTG CTG TGG TGG TGG GGCATG CGG CAG GCC ACC GTC 246 Ile Thr Arg Glu Leu Leu Trp Trp Trp Gly MetArg Gln Ala Thr Val 60 65 70 CAG CAA CTT GTG GAC CTC CTG TGC CGC CTG GAGCTC TAC CGG GCT GCC 294 Gln Gln Leu Val Asp Leu Leu Cys Arg Leu Glu LeuTyr Arg Ala Ala 75 80 85 CAG ATC ATC CTG AAC TGG AAA CCG GCT CCT GAA ATCAGG TGT CCC ATT 342 Gln Ile Ile Leu Asn Trp Lys Pro Ala Pro Glu Ile ArgCys Pro Ile 90 95 100 CCA GCC TTC CCT GAC TCT GTG AAG CCA GAA AAG CCTTTG GCA GCT TCT 390 Pro Ala Phe Pro Asp Ser Val Lys Pro Glu Lys Pro LeuAla Ala Ser 105 110 115 GTA AGA AAG GCT GAG GAT GAA CAG GAA GAG GGG CAGCCT GTG AGG ATG 438 Val Arg Lys Ala Glu Asp Glu Gln Glu Glu Gly Gln ProVal Arg Met 120 125 130 135 GCC ACC TTT CCA GGC CCA GGG TCC TCT CCA GCCAGA GCC CAC CAG CCG 486 Ala Thr Phe Pro Gly Pro Gly Ser Ser Pro Ala ArgAla His Gln Pro 140 145 150 GCC TTT CTC CAG CCT CCT GAA GAA GAT GCC CCTCAT TCC TTG AGA AGC 534 Ala Phe Leu Gln Pro Pro Glu Glu Asp Ala Pro HisSer Leu Arg Ser 155 160 165 GAC CTC CCC ACT TCG TCT GAT TCA AAG GAC TTCAGC ACC TCC ATT CCT 582 Asp Leu Pro Thr Ser Ser Asp Ser Lys Asp Phe SerThr Ser Ile Pro 170 175 180 AAG CAG GAA AAA CTT TTG AGC TTG GCT GGA GACAGC CTT TTC TGG AGT 630 Lys Gln Glu Lys Leu Leu Ser Leu Ala Gly Asp SerLeu Phe Trp Ser 185 190 195 GAG GCA GAC GTG GTC CAG GCA ACC GAT GAC TTCAAT CAA AAC CGC AAA 678 Glu Ala Asp Val Val Gln Ala Thr Asp Asp Phe AsnGln Asn Arg Lys 200 205 210 215 ATC AGC CAG GGG ACC TTT GCT GAC GTC TACAGA GGG CAC AGG CAC GGG 726 Ile Ser Gln Gly Thr Phe Ala Asp Val Tyr ArgGly His Arg His Gly 220 225 230 AAG CCA TTC GTC TTC AAG AAG CTC AGA GAGACA GCC TGT TCA AGT CCA 774 Lys Pro Phe Val Phe Lys Lys Leu Arg Glu ThrAla Cys Ser Ser Pro 235 240 245 GGA TCA ATC GAA AGA TTC TTC CAG GCA GAGTTG CAG ATT TGT CTT AGA 822 Gly Ser Ile Glu Arg Phe Phe Gln Ala Glu LeuGln Ile Cys Leu Arg 250 255 260 TGC TGC CAC CCC AAT GTC TTA CCT GTG CTGGGC TTC TGT GCT GCA AGA 870 Cys Cys His Pro Asn Val Leu Pro Val Leu GlyPhe Cys Ala Ala Arg 265 270 275 CAG TTT CAC AGC TTC ATC TAC CCC TAC ATGGCA AAT GGT TCC CTA CAG 918 Gln Phe His Ser Phe Ile Tyr Pro Tyr Met AlaAsn Gly Ser Leu Gln 280 285 290 295 GAC AGA CTG CAG GGT CAG GGT GGC TCGGAA CCC CTC CCC TGG CCC CAG 966 Asp Arg Leu Gln Gly Gln Gly Gly Ser GluPro Leu Pro Trp Pro Gln 300 305 310 CGT GTC AGC ATC TGC TCA GGG CTG CTCTGT GCC GTC GAG TAC CTG CAT 1014 Arg Val Ser Ile Cys Ser Gly Leu Leu CysAla Val Glu Tyr Leu His 315 320 325 GGT CTG GAG ATC ATC CAC AGC AAC GTCAAG AGC TCT AAT GTC TTG CTG 1062 Gly Leu Glu Ile Ile His Ser Asn Val LysSer Ser Asn Val Leu Leu 330 335 340 GAC CAA AAT CTC ACC CCC AAA CTT GCTCAC CCA ATG GCT CAT CTG TGT 1110 Asp Gln Asn Leu Thr Pro Lys Leu Ala HisPro Met Ala His Leu Cys 345 350 355 CCT GTC AAC AAA AGG TCA AAA TAC ACCATG ATG AAG ACT CAC CTG CTC 1158 Pro Val Asn Lys Arg Ser Lys Tyr Thr MetMet Lys Thr His Leu Leu 360 365 370 375 CGG ACG TCA GCC GCG TAT CTG CCAGAG GAT TTC ATC CGG GTG GGG CAG 1206 Arg Thr Ser Ala Ala Tyr Leu Pro GluAsp Phe Ile Arg Val Gly Gln 380 385 390 CTG ACA AAG CGA GTG GAC ATC TTCAGC TGT GGA ATA GTG TTG GCC GAG 1254 Leu Thr Lys Arg Val Asp Ile Phe SerCys Gly Ile Val Leu Ala Glu 395 400 405 GTC CTC ACG GGC ATC CCT GCA ATGGAT AAC AAC CGA AGC CCG GTT TAC 1302 Val Leu Thr Gly Ile Pro Ala Met AspAsn Asn Arg Ser Pro Val Tyr 410 415 420 CTG AAG GAC TTA CTC CTC AGT GAAATT CCA AGC AGC ACC GCC TCG CTC 1350 Leu Lys Asp Leu Leu Leu Ser Glu IlePro Ser Ser Thr Ala Ser Leu 425 430 435 TGC TCC AGG AAG ACG GGC GTG GAGAAC GTG ATG GCA AAG GAG ATC TGC 1398 Cys Ser Arg Lys Thr Gly Val Glu AsnVal Met Ala Lys Glu Ile Cys 440 445 450 455 CAG AAG TAC CTG GAG AAG GGCGCA GGG AGG CTT CCG GAG GAC TGC GCC 1446 Gln Lys Tyr Leu Glu Lys Gly AlaGly Arg Leu Pro Glu Asp Cys Ala 460 465 470 GAG GCC CTG GCC ACG GCT GCCTGC CTG TGC CTG CGG AGG CGT AAC ACC 1494 Glu Ala Leu Ala Thr Ala Ala CysLeu Cys Leu Arg Arg Arg Asn Thr 475 480 485 AGC CTG CAG GAG GTG TGT GGCTCT GTG GCT GCT GTG GAA GAG CGG CTC 1542 Ser Leu Gln Glu Val Cys Gly SerVal Ala Ala Val Glu Glu Arg Leu 490 495 500 CGA GGT CGG GAG ACG TTG CTCCCT TGG AGT GGG CTT TCT GAG GGT ACA 1590 Arg Gly Arg Glu Thr Leu Leu ProTrp Ser Gly Leu Ser Glu Gly Thr 505 510 515 GGC TCT TCT TCC AAC ACC CCAGAG GAA ACA GAC GAC GTT GAC AAT TCC 1638 Gly Ser Ser Ser Asn Thr Pro GluGlu Thr Asp Asp Val Asp Asn Ser 520 525 530 535 AGC CTT GAT GCC TCC TCCTCC ATG AGT GTG GCA CCC TGG GCA GGG GCT 1686 Ser Leu Asp Ala Ser Ser SerMet Ser Val Ala Pro Trp Ala Gly Ala 540 545 550 GCC ACC CCA CTT CTC CCCACA GAG AAT GGG GAA GGA AGG CTG CGG GTC 1734 Ala Thr Pro Leu Leu Pro ThrGlu Asn Gly Glu Gly Arg Leu Arg Val 555 560 565 ATC GTG GGA AGG GAG GCTGAC TCC TCC TCT GAG GCC TGT GTT GGC CTG 1782 Ile Val Gly Arg Glu Ala AspSer Ser Ser Glu Ala Cys Val Gly Leu 570 575 580 GAG CCT CCC CAG GAT GTTACA TAA 1806 Glu Pro Pro Gln Asp Val Thr 585 590 590 amino acids aminoacid linear protein 2 Met Ala Cys Tyr Ile Tyr Gln Leu Pro Ser Trp ValLeu Asp Asp Leu 1 5 10 15 Cys Arg Asn Met Asp Ala Leu Ser Glu Trp AspTrp Met Glu Phe Ala 20 25 30 Ser Tyr Val Ile Thr Asp Leu Thr Gln Leu ArgLys Ile Lys Ser Met 35 40 45 Glu Arg Val Gln Gly Val Ser Ile Thr Arg GluLeu Leu Trp Trp Trp 50 55 60 Gly Met Arg Gln Ala Thr Val Gln Gln Leu ValAsp Leu Leu Cys Arg 65 70 75 80 Leu Glu Leu Tyr Arg Ala Ala Gln Ile IleLeu Asn Trp Lys Pro Ala 85 90 95 Pro Glu Ile Arg Cys Pro Ile Pro Ala PhePro Asp Ser Val Lys Pro 100 105 110 Glu Lys Pro Leu Ala Ala Ser Val ArgLys Ala Glu Asp Glu Gln Glu 115 120 125 Glu Gly Gln Pro Val Arg Met AlaThr Phe Pro Gly Pro Gly Ser Ser 130 135 140 Pro Ala Arg Ala His Gln ProAla Phe Leu Gln Pro Pro Glu Glu Asp 145 150 155 160 Ala Pro His Ser LeuArg Ser Asp Leu Pro Thr Ser Ser Asp Ser Lys 165 170 175 Asp Phe Ser ThrSer Ile Pro Lys Gln Glu Lys Leu Leu Ser Leu Ala 180 185 190 Gly Asp SerLeu Phe Trp Ser Glu Ala Asp Val Val Gln Ala Thr Asp 195 200 205 Asp PheAsn Gln Asn Arg Lys Ile Ser Gln Gly Thr Phe Ala Asp Val 210 215 220 TyrArg Gly His Arg His Gly Lys Pro Phe Val Phe Lys Lys Leu Arg 225 230 235240 Glu Thr Ala Cys Ser Ser Pro Gly Ser Ile Glu Arg Phe Phe Gln Ala 245250 255 Glu Leu Gln Ile Cys Leu Arg Cys Cys His Pro Asn Val Leu Pro Val260 265 270 Leu Gly Phe Cys Ala Ala Arg Gln Phe His Ser Phe Ile Tyr ProTyr 275 280 285 Met Ala Asn Gly Ser Leu Gln Asp Arg Leu Gln Gly Gln GlyGly Ser 290 295 300 Glu Pro Leu Pro Trp Pro Gln Arg Val Ser Ile Cys SerGly Leu Leu 305 310 315 320 Cys Ala Val Glu Tyr Leu His Gly Leu Glu IleIle His Ser Asn Val 325 330 335 Lys Ser Ser Asn Val Leu Leu Asp Gln AsnLeu Thr Pro Lys Leu Ala 340 345 350 His Pro Met Ala His Leu Cys Pro ValAsn Lys Arg Ser Lys Tyr Thr 355 360 365 Met Met Lys Thr His Leu Leu ArgThr Ser Ala Ala Tyr Leu Pro Glu 370 375 380 Asp Phe Ile Arg Val Gly GlnLeu Thr Lys Arg Val Asp Ile Phe Ser 385 390 395 400 Cys Gly Ile Val LeuAla Glu Val Leu Thr Gly Ile Pro Ala Met Asp 405 410 415 Asn Asn Arg SerPro Val Tyr Leu Lys Asp Leu Leu Leu Ser Glu Ile 420 425 430 Pro Ser SerThr Ala Ser Leu Cys Ser Arg Lys Thr Gly Val Glu Asn 435 440 445 Val MetAla Lys Glu Ile Cys Gln Lys Tyr Leu Glu Lys Gly Ala Gly 450 455 460 ArgLeu Pro Glu Asp Cys Ala Glu Ala Leu Ala Thr Ala Ala Cys Leu 465 470 475480 Cys Leu Arg Arg Arg Asn Thr Ser Leu Gln Glu Val Cys Gly Ser Val 485490 495 Ala Ala Val Glu Glu Arg Leu Arg Gly Arg Glu Thr Leu Leu Pro Trp500 505 510 Ser Gly Leu Ser Glu Gly Thr Gly Ser Ser Ser Asn Thr Pro GluGlu 515 520 525 Thr Asp Asp Val Asp Asn Ser Ser Leu Asp Ala Ser Ser SerMet Ser 530 535 540 Val Ala Pro Trp Ala Gly Ala Ala Thr Pro Leu Leu ProThr Glu Asn 545 550 555 560 Gly Glu Gly Arg Leu Arg Val Ile Val Gly ArgGlu Ala Asp Ser Ser 565 570 575 Ser Glu Ala Cys Val Gly Leu Glu Pro ProGln Asp Val Thr 580 585 590 3459 base pairs nucleic acid double linearDNA (genomic) CDS 34..1908 3 GCAGGCGCGC CGGAGCCGGC CCCGTAGCGT GCC ATGGCC TGC TAC ATC TAC CAG 54 Met Ala Cys Tyr Ile Tyr Gln 1 5 CTG CCC TCCTGG GTG CTG GAC GAC CTG TGC CGC AAC ATG GAC GCG CTC 102 Leu Pro Ser TrpVal Leu Asp Asp Leu Cys Arg Asn Met Asp Ala Leu 10 15 20 AGC GAG TGG GACTGG ATG GAG TTC GCC TCC TAC GTG ATC ACA GAC CTG 150 Ser Glu Trp Asp TrpMet Glu Phe Ala Ser Tyr Val Ile Thr Asp Leu 25 30 35 ACC CAG CTG CGG AAGATC AAG TCC ATG GAG CGG GTG CAG GGT GTG AGC 198 Thr Gln Leu Arg Lys IleLys Ser Met Glu Arg Val Gln Gly Val Ser 40 45 50 55 ATC ACG CGG GAG CTGCTG TGG TGG TGG GGC ATG CGG CAG GCC ACC GTC 246 Ile Thr Arg Glu Leu LeuTrp Trp Trp Gly Met Arg Gln Ala Thr Val 60 65 70 CAG CAA CTT GTG GAC CTCCTG TGC CGC CTG GAG CTC TAC CGG GCT GCC 294 Gln Gln Leu Val Asp Leu LeuCys Arg Leu Glu Leu Tyr Arg Ala Ala 75 80 85 CAG ATC ATC CTG AAC TGG AAACCG GCT CCT GAA ATC AGG TGT CCC ATT 342 Gln Ile Ile Leu Asn Trp Lys ProAla Pro Glu Ile Arg Cys Pro Ile 90 95 100 CCA GCC TTC CCT GAC TCT GTGAAG CCA GAA AAG CCT TTG GCA GCT TCT 390 Pro Ala Phe Pro Asp Ser Val LysPro Glu Lys Pro Leu Ala Ala Ser 105 110 115 GTA AGA AAG GCT GAG GAT GAACAG GAA GAG GGG CAG CCT GTG AGG ATG 438 Val Arg Lys Ala Glu Asp Glu GlnGlu Glu Gly Gln Pro Val Arg Met 120 125 130 135 GCC ACC TTT CCA GGC CCAGGG TCC TCT CCA GCC AGA GCC CAC CAG CCG 486 Ala Thr Phe Pro Gly Pro GlySer Ser Pro Ala Arg Ala His Gln Pro 140 145 150 GCC TTT CTC CAG CCT CCTGAA GAA GAT GCC CCT CAT TCC TTG AGA AGC 534 Ala Phe Leu Gln Pro Pro GluGlu Asp Ala Pro His Ser Leu Arg Ser 155 160 165 GAC CTC CCC ACT TCG TCTGAT TCA AAG GAC TTC AGC ACC TCC ATT CCT 582 Asp Leu Pro Thr Ser Ser AspSer Lys Asp Phe Ser Thr Ser Ile Pro 170 175 180 AAG CAG GAA AAA CTT TTGAGC TTG GCT GGA GAC AGC CTT TTC TGG AGT 630 Lys Gln Glu Lys Leu Leu SerLeu Ala Gly Asp Ser Leu Phe Trp Ser 185 190 195 GAG GCA GAC GTG GTC CAGGCA ACC GAT GAC TTC AAT CAA AAC CGC AAA 678 Glu Ala Asp Val Val Gln AlaThr Asp Asp Phe Asn Gln Asn Arg Lys 200 205 210 215 ATC AGC CAG GGG ACCTTT GCT GAC GTC TAC AGA GGG CAC AGG CAC GGG 726 Ile Ser Gln Gly Thr PheAla Asp Val Tyr Arg Gly His Arg His Gly 220 225 230 AAG CCA TTC GTC TTCAAG AAG CTC AGA GAG ACA GCC TGT TCA AGT CCA 774 Lys Pro Phe Val Phe LysLys Leu Arg Glu Thr Ala Cys Ser Ser Pro 235 240 245 GGA TCA ATC GAA AGATTC TTC CAG GCA GAG TTG CAG ATT TGT CTT AGA 822 Gly Ser Ile Glu Arg PhePhe Gln Ala Glu Leu Gln Ile Cys Leu Arg 250 255 260 TGC TGC CAC CCC AATGTC TTA CCT GTG CTG GGC TTC TGT GCT GCA AGA 870 Cys Cys His Pro Asn ValLeu Pro Val Leu Gly Phe Cys Ala Ala Arg 265 270 275 CAG TTT CAC AGC TTCATC TAC CCC TAC ATG GCA AAT GGT TCC CTA CAG 918 Gln Phe His Ser Phe IleTyr Pro Tyr Met Ala Asn Gly Ser Leu Gln 280 285 290 295 GAC AGA CTG CAGGGT CAG GGT GGC TCG GAC CCC CTC CCC TGG CCC CAG 966 Asp Arg Leu Gln GlyGln Gly Gly Ser Asp Pro Leu Pro Trp Pro Gln 300 305 310 CGT GTC AGC ATCTGC TCA GGG CTG CTC TGT GCC GTC GAG TAC CTG CAT 1014 Arg Val Ser Ile CysSer Gly Leu Leu Cys Ala Val Glu Tyr Leu His 315 320 325 GGT CTG GAG ATCATC CAC AGC AAC GTC AAG AGC TCT AAT GTC TTG CTG 1062 Gly Leu Glu Ile IleHis Ser Asn Val Lys Ser Ser Asn Val Leu Leu 330 335 340 GAC CAA AAT CTCACC CCC AAA CTT GCT CAC CCA ATG GCT CAT CTG TGT 1110 Asp Gln Asn Leu ThrPro Lys Leu Ala His Pro Met Ala His Leu Cys 345 350 355 CCT GTC AAC AAAAGG TCA AAA TAC ACC ATG ATG AAG ACT CAC CTG CTC 1158 Pro Val Asn Lys ArgSer Lys Tyr Thr Met Met Lys Thr His Leu Leu 360 365 370 375 CGG ACG TCAGCC GCG TAT CTG CCA GAG GAT TTC ATC CGG GTG GGG CAG 1206 Arg Thr Ser AlaAla Tyr Leu Pro Glu Asp Phe Ile Arg Val Gly Gln 380 385 390 GTG ACA AAGCGA GTG GAC ATC TTC AGC TGT GGA ATA GTG TTG GCC GAG 1254 Val Thr Lys ArgVal Asp Ile Phe Ser Cys Gly Ile Val Leu Ala Glu 395 400 405 GTC CTC ACGGGC ATC CCT GCA ATG GAT AAC AAC CGA AGC CCG GTT TAC 1302 Val Leu Thr GlyIle Pro Ala Met Asp Asn Asn Arg Ser Pro Val Tyr 410 415 420 CTG AAG GACTTA CTC CTC AGT GAA ATT CCA AGC AGC ACC GCC TCG CTC 1350 Leu Lys Asp LeuLeu Leu Ser Glu Ile Pro Ser Ser Thr Ala Ser Leu 425 430 435 TGC TCC AGGAAG ACG GGC GTG GAG AAC GTG ATG GCA AAG GAG ATC TGC 1398 Cys Ser Arg LysThr Gly Val Glu Asn Val Met Ala Lys Glu Ile Cys 440 445 450 455 CAG AAGTAC CTG GAG AAG GGC GCA GGG AGG CTT CCG GAG GAC TGC GCC 1446 Gln Lys TyrLeu Glu Lys Gly Ala Gly Arg Leu Pro Glu Asp Cys Ala 460 465 470 GAG GCCCTG GCC ACG GCT GCC TGC CTG TGC CTG CGG AGG CGT AAC ACC 1494 Glu Ala LeuAla Thr Ala Ala Cys Leu Cys Leu Arg Arg Arg Asn Thr 475 480 485 AGC CTGCAG GAG GTG TGT GGC TCT GTG GCT GCT GTG GAA GAG CGG CTC 1542 Ser Leu GlnGlu Val Cys Gly Ser Val Ala Ala Val Glu Glu Arg Leu 490 495 500 CGA GGTCGG GAG ACG TTG CTC CCT TGG AGT GGG CTT TCT GAG GGT ACA 1590 Arg Gly ArgGlu Thr Leu Leu Pro Trp Ser Gly Leu Ser Glu Gly Thr 505 510 515 GGC TCTTCT TCC AAC ACC CCA GAG GAA ACA GAC GAC GTT GAC AAT TCC 1638 Gly Ser SerSer Asn Thr Pro Glu Glu Thr Asp Asp Val Asp Asn Ser 520 525 530 535 AGCCTT GAT GCC TCC TCC TCC ATG AGT GTG GCA CCC TGG GCA GGG GCT 1686 Ser LeuAsp Ala Ser Ser Ser Met Ser Val Ala Pro Trp Ala Gly Ala 540 545 550 GCCACC CCA CTT CTC CCC ACA GAG AAT GGG GAA GGA AGG CTG CGG GTC 1734 Ala ThrPro Leu Leu Pro Thr Glu Asn Gly Glu Gly Arg Leu Arg Val 555 560 565 ATCGTG GGA AGG GAG GCT GAC TCC TCC TCT GAG GCC TGT GTT GGC CTG 1782 Ile ValGly Arg Glu Ala Asp Ser Ser Ser Glu Ala Cys Val Gly Leu 570 575 580 GAGCCT CCC CAG GAT GTT ACA GAA ACT TCG TGG CAA ATT GAG ATC AAT 1830 Glu ProPro Gln Asp Val Thr Glu Thr Ser Trp Gln Ile Glu Ile Asn 585 590 595 GAGGCC AAA AGG AAA CTG ATG GAG AAT ATT CTG CTC TAC AAA GAG GAA 1878 Glu AlaLys Arg Lys Leu Met Glu Asn Ile Leu Leu Tyr Lys Glu Glu 610 605 610 615AAA GTG GAC AGC ATT GAG CTC TTT GGC CCC TGATGACCGG AACACAGCTG 1928 LysVal Asp Ser Ile Glu Leu Phe Gly Pro 620 625 AGGACCCTTG TCCTCAGTTGGAAAGATGAG CATCAGATCA AGAAAAAGGT CTGAGGCA 1988 ATCCAAGATC TGCCAGGAAACACACAACAA AACATCTGCT GTCCTGGGTG GGAGGGAA 2048 TTCATTTCAC TGGAATGAGTTGGGAGAGAA AGGCCCTCAG CTTTTAGAGA CACAAAAA 2108 CATGAAGTCT CTTCCTTTCTGGGCTTTGTT AGTCAGAGCA GGGGATCAGA GGAGACTG 2168 GCAGAAACCC TGCACACGGGCCCAGGATGT GGCTGATTTT GTGGTTCCGG GGAGTATG 2228 ATGATAATCA CCCCCAGCAGATTCCATTAC CTCAGCAGCT CTTGTTCCCC CGCCACTG 2288 AGTTCTGCAA TGCCATAGCATTTTCCAGAG CTAAGATCTC TGGGTTGTAT TTGCTGAC 2348 CCTGCAAGCT TGCATGCTCTGAAAGATTTT TTTAGTTTTT AATTTTTTTG TAAAAATG 2408 GTCTCGCTTT GTTGGCGCAATCCTCCCACC TCAGACTCCC AAAGTGCTGG AATTACAT 2468 GGAACCACTG TGCCTGGCCTGGAAAACTTC CAACTTGTGT TCTCAGTGCA GTTCTGAC 2528 ACCTCTCTGG GCCTCAGGTTCTACAAATGC CAGACACCTA GCGAAGAGCT CTGCAGGC 2588 TCCACTGCCT GTATTGGAAATCTTGCAATT CACATAATTA TTCAGTCACT GCCTGGTA 2648 TTTATCTTCC CATCCCATTAATGTTAGTGT TTTTTAATGG AGCTTTTATT CTGAGAAT 2708 GTGTTCGTCT GTTTGTTTGTTTTTTGAGAC AGAGTCTCAC TTTGTCACCC AGGCTGGA 2768 GCAGTGGCAC GATCTCAGCTCACTGCAAGC TGTGCCTCTC AGGTTTCAAG TGATTCTC 2828 GCCTCAGCCT CCTGAGTAGATGGGACTGTA GGCACCTGCC ACTATGCCTG GCTAATTT 2888 GTGTTTTTAG TAGAGACAGGGTTTCACCAT ATTGGCCAGG CTGGTCTCGA ACTACTGA 2948 TCGTGATCTG CCCGCCTTGGCCTATCAAAG TGTTGGGATT ACAGGCTTGA GCCACCGC 3008 CCGGCCGAGA ATATGTGTTGTTATTTATGA CTGGATTATG AAGAATCAGG AGAATGCA 3068 TCATGTCTGA TTCTGCTGCTAATTAAGTCA ATCATTTAAT TTTTGGGACC TCAGTTTC 3128 TGTAAGTAAA ATAACACCTGCTTGTTCTTC ATCCCTGGGC TGTTGGGAGG AACAGATG 3188 ACAGTGGCTA TAGAAGCACTTGGAAAATGC ACTTGTCCTG TTTTGTAAAA TAAAAAGG 3248 TTAAATGTGT ATTTCTGCCATGTACCTAAT GATTATTCAG TGCGTATATA TCTGAAAA 3308 CATGTTGCAA ATCTTTCTGTGAAACAGATG CTATTTTAAA TTCACTGGGA GAAATATC 3368 ATTTAAAGTA ATCTATAGTAATTTCTTTTT ATATAATAAA AATATATTTG TAAAGTCG 3428 AAAAAAAAAA AAAAAAAAAAAAAAAAAAAA A 3459 625 amino acids amino acid linear protein 4 Met AlaCys Tyr Ile Tyr Gln Leu Pro Ser Trp Val Leu Asp Asp Leu 1 5 10 15 CysArg Asn Met Asp Ala Leu Ser Glu Trp Asp Trp Met Glu Phe Ala 20 25 30 SerTyr Val Ile Thr Asp Leu Thr Gln Leu Arg Lys Ile Lys Ser Met 35 40 45 GluArg Val Gln Gly Val Ser Ile Thr Arg Glu Leu Leu Trp Trp Trp 50 55 60 GlyMet Arg Gln Ala Thr Val Gln Gln Leu Val Asp Leu Leu Cys Arg 65 70 75 80Leu Glu Leu Tyr Arg Ala Ala Gln Ile Ile Leu Asn Trp Lys Pro Ala 85 90 95Pro Glu Ile Arg Cys Pro Ile Pro Ala Phe Pro Asp Ser Val Lys Pro 100 105110 Glu Lys Pro Leu Ala Ala Ser Val Arg Lys Ala Glu Asp Glu Gln Glu 115120 125 Glu Gly Gln Pro Val Arg Met Ala Thr Phe Pro Gly Pro Gly Ser Ser130 135 140 Pro Ala Arg Ala His Gln Pro Ala Phe Leu Gln Pro Pro Glu GluAsp 145 150 155 160 Ala Pro His Ser Leu Arg Ser Asp Leu Pro Thr Ser SerAsp Ser Lys 165 170 175 Asp Phe Ser Thr Ser Ile Pro Lys Gln Glu Lys LeuLeu Ser Leu Ala 180 185 190 Gly Asp Ser Leu Phe Trp Ser Glu Ala Asp ValVal Gln Ala Thr Asp 195 200 205 Asp Phe Asn Gln Asn Arg Lys Ile Ser GlnGly Thr Phe Ala Asp Val 210 215 220 Tyr Arg Gly His Arg His Gly Lys ProPhe Val Phe Lys Lys Leu Arg 225 230 235 240 Glu Thr Ala Cys Ser Ser ProGly Ser Ile Glu Arg Phe Phe Gln Ala 245 250 255 Glu Leu Gln Ile Cys LeuArg Cys Cys His Pro Asn Val Leu Pro Val 260 265 270 Leu Gly Phe Cys AlaAla Arg Gln Phe His Ser Phe Ile Tyr Pro Tyr 275 280 285 Met Ala Asn GlySer Leu Gln Asp Arg Leu Gln Gly Gln Gly Gly Ser 290 295 300 Asp Pro LeuPro Trp Pro Gln Arg Val Ser Ile Cys Ser Gly Leu Leu 305 310 315 320 CysAla Val Glu Tyr Leu His Gly Leu Glu Ile Ile His Ser Asn Val 325 330 335Lys Ser Ser Asn Val Leu Leu Asp Gln Asn Leu Thr Pro Lys Leu Ala 340 345350 His Pro Met Ala His Leu Cys Pro Val Asn Lys Arg Ser Lys Tyr Thr 355360 365 Met Met Lys Thr His Leu Leu Arg Thr Ser Ala Ala Tyr Leu Pro Glu370 375 380 Asp Phe Ile Arg Val Gly Gln Val Thr Lys Arg Val Asp Ile PheSer 385 390 395 400 Cys Gly Ile Val Leu Ala Glu Val Leu Thr Gly Ile ProAla Met Asp 405 410 415 Asn Asn Arg Ser Pro Val Tyr Leu Lys Asp Leu LeuLeu Ser Glu Ile 420 425 430 Pro Ser Ser Thr Ala Ser Leu Cys Ser Arg LysThr Gly Val Glu Asn 435 440 445 Val Met Ala Lys Glu Ile Cys Gln Lys TyrLeu Glu Lys Gly Ala Gly 450 455 460 Arg Leu Pro Glu Asp Cys Ala Glu AlaLeu Ala Thr Ala Ala Cys Leu 465 470 475 480 Cys Leu Arg Arg Arg Asn ThrSer Leu Gln Glu Val Cys Gly Ser Val 485 490 495 Ala Ala Val Glu Glu ArgLeu Arg Gly Arg Glu Thr Leu Leu Pro Trp 500 505 510 Ser Gly Leu Ser GluGly Thr Gly Ser Ser Ser Asn Thr Pro Glu Glu 515 520 525 Thr Asp Asp ValAsp Asn Ser Ser Leu Asp Ala Ser Ser Ser Met Ser 530 535 540 Val Ala ProTrp Ala Gly Ala Ala Thr Pro Leu Leu Pro Thr Glu Asn 545 550 555 560 GlyGlu Gly Arg Leu Arg Val Ile Val Gly Arg Glu Ala Asp Ser Ser 565 570 575Ser Glu Ala Cys Val Gly Leu Glu Pro Pro Gln Asp Val Thr Glu Thr 580 585590 Ser Trp Gln Ile Glu Ile Asn Glu Ala Lys Arg Lys Leu Met Glu Asn 595600 605 Ile Leu Leu Tyr Lys Glu Glu Lys Val Asp Ser Ile Glu Leu Phe Gly610 615 620 Pro 625 712 amino acids amino acid single Not Relevantprotein 5 Met Ala Gly Gly Pro Gly Pro Gly Glu Pro Ala Ala Pro Gly Ala Gl1 5 10 15 His Phe Leu Tyr Glu Val Pro Pro Trp Val Met Cys Arg Phe Tyr Ly20 25 30 Val Met Asp Ala Leu Glu Pro Ala Asp Trp Cys Gln Phe Ala Ala Le35 40 45 Ile Val Arg Asp Gln Thr Glu Leu Arg Leu Cys Glu Arg Ser Gly Gl50 55 60 Arg Thr Ala Ser Val Leu Trp Pro Trp Ile Asn Arg Asn Ala Arg Va65 70 75 80 Ala Asp Leu Val His Ile Leu Thr His Leu Gln Leu Leu Arg AlaAr 85 90 95 Asp Ile Ile Thr Ala Trp His Pro Pro Ala Pro Leu Pro Ser ProGl 100 105 110 Thr Thr Ala Pro Arg Pro Ser Ser Ile Pro Ala Pro Ala GluAla Gl 115 120 125 Ala Trp Ser Pro Arg Lys Leu Pro Ser Ser Ala Ser ThrPhe Leu Se 130 135 140 Pro Ala Phe Pro Gly Ser Gln Thr His Ser Gly ProGlu Leu Gly Le 145 150 155 160 Val Pro Ser Pro Ala Ser Leu Trp Pro ProPro Pro Ser Pro Ala Pr 165 170 175 Ser Ser Thr Lys Pro Gly Pro Glu SerSer Val Ser Leu Leu Gln Gl 180 185 190 Ala Arg Pro Ser Pro Phe Cys TrpPro Leu Cys Glu Ile Ser Arg Gl 195 200 205 Thr His Asn Phe Ser Glu GluLeu Lys Ile Gly Glu Gly Gly Phe Gl 210 215 220 Cys Val Tyr Arg Ala ValMet Arg Asn Thr Val Tyr Ala Val Lys Ar 225 230 235 240 Leu Lys Glu AsnAla Asp Leu Glu Trp Thr Ala Val Lys Gln Ser Ph 245 250 255 Leu Thr GluVal Glu Gln Leu Ser Arg Phe Arg His Pro Asn Ile Va 260 265 270 Asp PheAla Gly Tyr Cys Ala Gln Asn Gly Phe Tyr Cys Leu Val Ty 275 280 285 GlyPhe Leu Pro Asn Gly Ser Leu Glu Asp Arg Leu His Cys Gln Th 290 295 300Gln Ala Cys Pro Pro Leu Ser Trp Pro Gln Arg Leu Asp Ile Leu Le 305 310315 320 Gly Thr Ala Arg Ala Ile Gln Phe Leu His Gln Asp Ser Pro Ser Le325 330 335 Ile His Gly Asp Ile Lys Ser Ser Asn Val Leu Leu Asp Glu ArgLe 340 345 350 Thr Pro Lys Leu Gly Asp Phe Gly Leu Ala Arg Phe Ser ArgPhe Al 355 360 365 Gly Ser Ser Pro Ser Gln Ser Ser Met Val Ala Arg ThrGln Thr Va 370 375 380 Arg Gly Thr Leu Ala Tyr Leu Pro Glu Glu Tyr IleLys Thr Gly Ar 385 390 395 400 Leu Ala Val Asp Thr Asp Thr Phe Ser PheGly Val Val Val Leu Gl 405 410 415 Thr Leu Ala Gly Gln Arg Ala Val LysThr His Gly Ala Arg Thr Ly 420 425 430 Tyr Leu Lys Asp Leu Val Glu GluGlu Ala Glu Glu Ala Gly Val Al 435 440 445 Leu Arg Ser Thr Gln Ser ThrLeu Gln Ala Gly Leu Ala Ala Asp Al 450 455 460 Trp Ala Ala Pro Ile AlaMet Gln Ile Tyr Lys Lys His Leu Asp Pr 465 470 475 480 Arg Pro Gly ProCys Pro Pro Glu Leu Gly Leu Gly Leu Gly Gln Le 485 490 495 Ala Cys CysCys Leu His Arg Arg Ala Lys Arg Arg Pro Pro Met Th 500 505 510 Gln ValTyr Glu Arg Leu Glu Lys Leu Gln Ala Val Val Ala Gly Va 515 520 525 ProGly His Leu Glu Ala Ala Ser Cys Ile Pro Pro Ser Pro Gln Gl 530 535 540Asn Ser Tyr Val Ser Ser Thr Gly Arg Ala His Ser Gly Ala Ala Pr 545 550555 560 Trp Gln Pro Leu Ala Ala Pro Ser Gly Ala Ser Ala Gln Ala Ala Gl565 570 575 Gln Leu Gln Arg Gly Pro Asn Gln Pro Val Glu Ser Asp Glu SerLe 580 585 590 Gly Gly Leu Ser Ala Ala Leu Arg Ser Trp His Leu Thr ProSer Cy 595 600 605 Pro Leu Asp Pro Ala Pro Leu Arg Glu Ala Gly Cys ProGln Gly As 610 615 620 Thr Ala Gly Glu Ser Ser Trp Gly Ser Gly Pro GlySer Arg Pro Th 625 630 635 640 Ala Val Glu Gly Leu Ala Leu Gly Ser SerAla Ser Ser Ser Ser Gl 645 650 655 Pro Pro Gln Ile Ile Ile Asn Pro AlaArg Gln Lys Met Val Gln Ly 660 665 670 Leu Ala Leu Tyr Glu Asp Gly AlaLeu Asp Ser Leu Gln Leu Leu Se 675 680 685 Ser Ser Ser Leu Pro Gly LeuGly Leu Glu Gln Asp Arg Gln Gly Pr 690 695 700 Glu Glu Ser Asp Glu PheGln Ser 705 710 501 amino acids amino acid single Not Relevant protein 6Met Ser Gly Val Gln Thr Ala Glu Ala Glu Ala Gln Ala Gln Asn Gl 1 5 10 15Ala Asn Gly Asn Arg Thr Arg Ser Arg Ser His Leu Asp Asn Thr Me 20 25 30Ala Ile Arg Leu Leu Pro Leu Pro Val Arg Ala Gln Leu Cys Ala Hi 35 40 45Leu Asp Ala Leu Asp Val Trp Gln Gln Leu Ala Thr Ala Val Lys Le 50 55 60Tyr Pro Asp Gln Val Glu Gln Ile Ser Ser Gln Lys Gln Arg Gly Ar 65 70 7580 Ser Ala Ser Asn Glu Phe Leu Asn Ile Trp Gly Gly Gln Tyr Asn Hi 85 9095 Thr Val Gln Thr Leu Phe Ala Leu Phe Lys Lys Leu Lys Leu His As 100105 110 Ala Met Arg Leu Ile Lys Asp Tyr Val Ser Glu Asp Leu His Lys Ty115 120 125 Ile Pro Arg Ser Val Pro Thr Ile Ser Glu Leu Arg Ala Ala ProAs 130 135 140 Ser Ser Ala Lys Val Asn Asn Gly Pro Pro Phe Pro Ser SerSer Gl 145 150 155 160 Val Ser Asn Ser Asn Asn Asn Arg Thr Ser Thr ThrAla Thr Glu Gl 165 170 175 Ile Pro Ser Leu Glu Ser Leu Gly Asn Ile HisIle Ser Thr Val Gl 180 185 190 Arg Ala Ala Glu Ser Leu Leu Glu Ile AspTyr Ala Glu Leu Glu As 195 200 205 Ala Thr Asp Gly Trp Ser Pro Asp AsnArg Leu Gly Gln Gly Gly Ph 210 215 220 Gly Asp Val Tyr Arg Gly Lys TrpLys Gln Leu Asp Val Ala Ile Ly 225 230 235 240 Val Met Asn Tyr Arg SerPro Asn Ile Asp Gln Lys Met Val Glu Le 245 250 255 Gln Gln Ser Tyr AsnGlu Leu Lys Tyr Leu Asn Ser Ile Arg His As 260 265 270 Asn Ile Leu AlaLeu Tyr Gly Tyr Ser Ile Lys Gly Gly Lys Pro Cy 275 280 285 Leu Val TyrGln Leu Met Lys Gly Gly Ser Leu Glu Ala Arg Leu Ar 290 295 300 Ala HisLys Ala Gln Asn Pro Leu Pro Ala Leu Thr Trp Gln Gln Ar 305 310 315 320Phe Ser Ile Ser Leu Gly Thr Ala Arg Gly Ile Tyr Phe Leu His Th 325 330335 Ala Arg Gly Thr Pro Leu Ile His Gly Asp Ile Lys Pro Ala Asn Il 340345 350 Leu Leu Asp Gln Cys Leu Gln Pro Lys Ile Gly Asp Phe Gly Leu Va355 360 365 Arg Glu Gly Pro Lys Ser Leu Asp Ala Val Val Glu Val Asn LysVa 370 375 380 Phe Gly Thr Lys Ile Tyr Leu Pro Pro Glu Phe Arg Asn PheArg Gl 385 390 395 400 Leu Ser Thr Gly Val Asp Val Tyr Ser Phe Gly IleVal Leu Leu Gl 405 410 415 Val Phe Thr Gly Arg Gln Val Thr Asp Arg ValPro Glu Asn Glu Th 420 425 430 Lys Lys Asn Leu Leu Asp Tyr Val Lys GlnGln Trp Arg Gln Asn Ar 435 440 445 Met Glu Leu Leu Glu Lys His Leu AlaAla Pro Met Gly Lys Glu Le 450 455 460 Asp Met Cys Met Cys Ala Ile GluAla Gly Leu His Cys Thr Ala Le 465 470 475 480 Asp Pro Gln Asp Arg ProSer Met Asn Ala Val Leu Lys Arg Phe Gl 485 490 495 Pro Phe Val Thr Asp500 265 base pairs nucleic acid double linear cDNA 7 GGCACGAGCACCTTTCCAGG CCCAGGGTCC TNTCCAGCCA GAGCCCACCA GCCGGCCTTT 60 CTCCAGCCTCCTGAAGAAGA TNNCCCTCAT TCCTTGAGAA GCGACCTCCC CACTTCGTC 120 GNTTCAAAGGACTTCAGCAC CTCCATTCCT AAGCAGGAAA AACTTTTGAG CTTGGCTGG 180 GACAGATGNTTCTGGTGTGA GGCAGACGTG GTCCAGTCAA CCGATGACTT GANTNNTAA 240 CGCAGAATCAGNCAGGGGAC CTTTG 265 294 base pairs nucleic acid double linear cDNA 8AACGTCAAGA GCTCTAATNT CTTGCTGGAC CAAAATCTNA CCCCCAAACT TGCTCACCCA 60ATGGCTCATC TGTGTCCTGT NAACAAAAGG TCAAAATACA CCATGATGAT GACTCACCT 120GCTCCGGAAC GTCAGCCGCG TATCTCCCAG NGGATTTNAT CCGGAGTGGG GCAGCTGAA 180AAAGCGAGTG GACATCTTCA GCTGTGGAAT AGTGTTGGAC GAGGTNCTCA CGGGGNATC 240CTGTCAATGG GTTAACANCC GAAGCCCGGT TTACCTGAAG GNACTTAATT NCTC 294 330 basepairs nucleic acid double linear cDNA 9 CNCTAATGTN TTNCTGGACC AAAATNTCACCCCCAAACTT CCTCACCCAA TGGCTCATCT 60 NTTTCCTGTC AACAAAAGGT CAAAATACACCATGATGAAG ACTCACCTGC TCCGGACGT 120 AGCCGCGTAT CTGCCCAGAG GATTTCATCCGNGTGGGGCA GCTGACAAAG CGAGTGGAC 180 TCTTCAGCTG TGGAATAGTA AGAGTGTCCTGCTCTGCGTA GAAGTGGGGC CCACCTTGA 240 TTTGTCCTTC CCACGGTTCC TTTGTNAATCACAGGATACG GTAGAGNCAC ACAGACAGG 300 TCCNNCAAGT NACAACAGGG GCTGTACAAA 330499 base pairs nucleic acid double linear cDNA 10 AATTCGGCAN AGNATGGAGTTCGCCTCCTA CGTGATCACA GACCTGACCC AGCTGCGGAA 60 GATCAAGTCC ATGGAGCGGGTGCAGGGTGT GAGCATCACG CGGGGNGCTG CTGTGGTGG 120 GGGGCATGCG GCAGGCCACCGTCCAGCAAC TTGTGGGACC TCCTGTGCCG CCTGGGAGC 180 CTACCGGGNT GCCCAGATCATCCTGGAACT TGTGGACACA AGACTTCTCA CATCTGAGA 240 GGCCCCTCTG TGCCCCTACATGCACATTGG CAGACAGCAA GAAGGGAAAA AGAGGGAAA 300 AGGGAAACCG GCTNCTGGAAATCAGGTGTN CCCATTTCCA GNCTTTCCCT GAATTCTNT 360 GAAGGCCAGA AAAAGCCTTTTGGCAAGGTT TTTGTTAAGN AAAGGNTNNA GGTTGAACC 420 GGAAGAGGGG GCAGNCTTTNAAGGNNTGGG CCACTTTTTN CAGGGCCCCA GGGGTCCTT 480 TNCAGCCCGN GGNCCAACC 499413 base pairs nucleic acid double linear cDNA 11 GGGCACNAGG NGGGTCATCGTGGGAAGGGA AGGCTGACTC CTCCTCTGAA GGACTGTTTT 60 GGANCTAGAG CGTCCCCAGGNTGTTACAGA AACTTCGTGG NNAAATTGAG AATCAATGA 120 GGCAAAAGGA AACTGATGGNGAATATTCTG CTCTACAANG AGGGAGAAAG TGGNCAGNA 180 TGAGCTNTTT GGCCCCTAATGACCGGAACA GAGCTGAGGN NCCTTGTCCT CAGTTGGAA 240 GATGAGCATC AGATCAAGAAAAAGGTCTGA GGTAGANTNC AAGATCTGNC ANGNAACAN 300 CANCANGACA TCTGCTGTCCTNGGTGGGGG GGAAACTTAT TTACTGGAAT GAGTTTGGA 360 AGAAAGGCCC TCAANATTTTGGTGGCACAA ANATCCATGA AGGNTATTCG ATN 413 665 base pairs nucleic aciddouble linear cDNA 12 AGAGAAGCCG CAGCCCGCAG TGTCCGACCC AGTCGTCCCGCGCCGGAGCC GGCCCCGTAG 60 CGTGCCATGG CCTGCTACAT CTACCAGCTG CCCTCCTGGGTGCTGGACGA CCTGTGCCG 120 AACATGGACG CGCTCAGGAG TGGGACTGGA TGGAGTTCGGTGAGTGCGGC CCGGGGAGG 180 GAGGGGACCA GGGCGACCGG AGCCCCCAGC GATCCCGCCTGGAGCGGCCG CCAAGCTCC 240 TCGGGCACCC GGGTTCAGCG GGTCCCGATC CGAGGGCGTGCGAGCTGAGC CTTCCTGGA 300 CGGGTTCCGC CGCGGACCTT CGGCCTGTTC ACCTGAAGGTGCCGGTGGTC TCTGAGGAC 360 TCTGTTCGAC GAGCCAGGGG CCGCCGCCAC TGCGCTCTGAGTCCAGAGAA CGGTGGGTA 420 GGGGGCCCTC CTGTCAGCGC TGCTGGCTCG GTGACGTCCCCAGGTGGCCT CTCATCCAG 480 CCACAACAGC CTGCAAAGTG CGAGCCTCGA CCCTGTAGGGACCCACGGTG CTGTCACTT 540 TTGGGGGTGT GTGTGTGTGT GTGTGTGTGG TGTGTTTAGTTTTAGTGTAT ATTAGAAGG 600 TCTATGATTT AACATATATA TATATATTGA AACAGAGCAAGATTCTGTCT CAAAAAAAA 660 AAAAA 665 327 base pairs nucleic acid doublelinear cDNA 13 AGGGAAACTG ATGGNGAATA TTCTGCTCTA CAAAGAGGNA AAAAGTGGACAGCATTGAGC 60 TCTTTGGCCC CTGATGACCG GAACACAGCT GAGGACCCTT GTCCTCAGTTGGAAAGATG 120 GCATCAGATC AAGAAAAAGG TCTGAGGCAG AATCCAAGAT CTGCCAGGAAACACACAAC 180 AAACATCTGC TGTCCTGGGT GGGAGGGAAA CTTCATTTCA CTGGAATGAGTTGGGAGAG 240 AAGGCCCTCA GCTTTTNGGG ANACAAAATT CCNTGAGGTT TTTCCCTTCNTGGTTTNTA 300 GTAAGGGCAG GGTTAAGGGG TTTAGGA 327 479 base pairs nucleicacid double linear cDNA 14 AGTATTAAGG CCAGAGAGTG CAACTCACAC GGATGGAAACTGCTCAGGAG CGTGATGGGC 60 CCCACCCAAG GAGGGCCTGG AGTTACTCAC AGTTCAGGATGATCTGGGCA GCCCGGTAG 120 GCTCCAGGCG GCACAGGAGG TCCACAAGTT GCTGGACGGTGGCCTGCCGC ATGCCCCAC 180 ACCACAGCAG CTCCCGCGTG ATGCTCACAC CCTGCACCCGCTCCATGGAC TTGATCTTC 240 GCAGCTGGGT CAGGTCTGTG ATCACGTAGG AGGCTGGAAGGGACAGAGAG AACTCTGCT 300 AGAGTCAGAG AGGCAGTCCC TCTAGGACAG GTCCCCACACTAAGCCCCTA GCTTGGGTT 360 TTCCAGGACA TCCTCCCCAA CCAACCGCCT CCACACTGGAAACACCACCA TTAAGCTGA 420 GTCCACAGGT GGCCAAGTTA CAACGCTGAC TCTGCTGGGCACCCATGGGG TCCAGTACA 479

What is claimed is:
 1. An isolated nucleic acid molecule comprising apolynucleotide having a nucleotide sequence at least 95% identical to asequence selected from the group consisting of: (a) a nucleotidesequence encoding a polypeptide comprising amino acids from about 1 toabout 590 in SEQ ID NO:2; (b) a nucleotide sequence encoding apolypeptide comprising amino acids from about 2 to about 590 in SEQ IDNO:2; (c) a nucleotide sequence encoding a polypeptide comprising aminoacids from about 1 to about 625 in SEQ ID NO:4; (d) a nucleotidesequence encoding a polypeptide comprising amino acids from about 2 toabout 625 in SEQ ID NO:4; (e) a nucleotide sequence encoding apolypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit NO. 209340; and (f) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), (c), (d),or (e).
 2. An isolated nucleic acid molecule comprising a polynucleotidewhich hybridizes under stringent hybridization conditions to apolynucleotide having a nucleotide sequence identical to a nucleotidesequence in (a), (b), (c), (d), (e), or (f) of claim 1 wherein saidpolynucleotide which hybridizes does not hybridize under stringenthybridization conditions to a polynucleotide having a nucleotidesequence consisting of only A residues or of only T residues.
 3. Anisolated nucleic acid molecule comprising a polynucleotide which encodesthe amino acid sequence of an epitope-bearing portion of an IRAK-2polypeptide having an amino acid sequence in (a), (b), (c), (d), or (e)of claim
 1. 4. The isolated nucleic acid molecule of claim 3, whichencodes an epitope-bearing portion of an IRAK-2 polypeptide selectedfrom the group consisting of: a polypeptide comprising amino acidresidues from about 96 to about 193 in SEQ ID NO:2 or SEQ ID NO:4; apolypeptide comprising amino acid residues from about 207 to about 254in SEQ ID NO:2 OR SEQ ID NO:4; a polypeptide comprising amino acidresidues from about 293 to about 316 in SEQ ID NO:2 or SEQ ID NO: 4; apolypeptide comprising amino acid residues from about 416 to about 472in SEQ ID NO:2 or SEQ ID NO:4; a polypeptide comprising amino acidresidues from about 487 to about 541 in SEQ ID NO:2 or SEQ ID NO:4; anda polypeptide comprising amino acid residues from about 559 to about 619in SEQ ID NO:4.
 5. An isolated nucleic acid molecule, comprising apolynucleotide having a sequence selected from the group consisting of:(a) the nucleotide sequence of a fragment of the sequences shown in SEQID NO:1 or SEQ ID NO:3, wherein said fragment comprises at least 50contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:3, provided that saidisolated nucleic acid molecule is not SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ IDNO:14, or any subfragment thereof; and (b) a nucleotide sequencecomplementary to a nucleotide sequence in (a).
 6. A method for making arecombinant vector comprising inserting an isolated nucleic acidmolecule of claim 1 into a vector.
 7. A recombinant vector produced bythe method of claim
 6. 8. A method of making a recombinant host cellcomprising introducing the recombinant vector of claim 7 into a hostcell.
 9. A recombinant host cell produced by the method of claim
 8. 10.A recombinant method for producing an IRAK-2 polypeptide, comprisingculturing the recombinant host cell of claim 9 under conditions suchthat said polypeptide is expressed and recovering said polypeptide. 11.An isolated IRAK-2 polypeptide having an amino acid sequence at least95% identical to a sequence selected from the group consisting of: (a)amino acids from about 1 to about 590 in SEQ ID NO:2; (b) amino acidsfrom about 2 to about 590 in SEQ ID NO:2; (c) amino acids from about 1to about 625 in SEQ ID NO:4; (d) amino acids from about 2 to about 625in SEQ ID NO:4; (e) the amino acid sequence of the IRAK-2 polypeptidehaving the amino acid sequence encoded by the cDNA clone contained inATCC Deposit No. 209340; and (f) the amino acid sequence of anepitope-bearing portion of any one of the polypeptides of (a), (b), (c),(d), or (e).
 12. An isolated polypeptide comprising an epitope-bearingportion of the IRAK-2 protein, wherein said portion is selected from thegroup consisting of: a polypeptide comprising amino acid residues fromabout 96 to about 193 in SEQ ID NO:2 or SEQ ID NO:4; a polypeptidecomprising amino acid residues from about 207 to about 254 in SEQ IDNO:2 OR SEQ ID NO:4; a polypeptide comprising amino acid residues fromabout 293 to about 316 in SEQ ID NO:2 or SEQ ID NO: 4; a polypeptidecomprising amino acid residues from about 416 to about 472 in SEQ IDNO:2 or SEQ ID NO:4; a polypeptide comprising amino acid residues fromabout 487 to about 541 in SEQ ID NO:2 or SEQ ID NO:4; and a polypeptidecomprising amino acid residues from about 559 to about 619 in SEQ IDNO:4.
 13. The isolated polypeptide of claim 11, which is produced orcontained in a recombinant host cell.
 14. The isolated polypeptide ofclaim 13, wherein said recombinant host cell is mammalian.
 15. Anisolated nucleic acid molecule comprising a polynucleotide encoding anIRAK-2 polypeptide wherein, except for one to fifty conservative aminoacid substitutions, said polypeptide has a sequence selected from thegroup consisting of: (a) a nucleotide sequence encoding a polypeptidecomprising amino acids from about 1 to about 590 in SEQ ID NO:2; (b) anucleotide sequence encoding a polypeptide comprising amino acids fromabout 2 to about 590 in SEQ ID NO:2; (c) a nucleotide sequence encodinga polypeptide comprising amino acids from about 1 to about 625 in SEQ IDNO:4; (d) a nucleotide sequence encoding a polypeptide comprising aminoacids from about 2 to about 625 in SEQ ID NO:4; (e) a nucleotidesequence encoding a polypeptide having the amino acid sequence encodedby the cDNA clone contained in ATCC Deposit No. 209340; and (f) anucleotide sequence complementary to any of the nucleotide sequences in(a), (b), (c), (d), or (e).
 16. An isolated IRAK-2 polypeptide wherein,except for one to fifty conservative amino acid substitutions, saidpolypeptide has a sequence selected from the group consisting of: (a)amino acids from about 1 to about 590 in SEQ ID NO:2; (b) amino acidsfrom about 2 to about 590 in SEQ ID NO:2; (c) amino acids from about 1to about 625 in SEQ ID NO:4; (d) amino acids from about 2 to about 625in SEQ ID NO:4; (e) the amino acid sequence of the IRAK-2 polypeptidehaving the amino acid sequence encoded by the cDNA clone contained inATCC Deposit No. 209340; and (f) the amino acid sequence of anepitope-bearing portion of any one of the polypeptides of (a), (b), (c),(d), or (e).
 17. A method for determining if a compound of interest isan agonist or antagonist of the IRAK-2 pathway, comprising: (a)transfecting cells which express IRAK-2 with a construct comprising areporter gene operably linked to a promoter which is activated by NF-kB;(b) contacting said cells with a compound of interest; (c) assaying saidreporter gene expression; and (d) comparing said reporter geneexpression to a standard reporter gene expression, said standard beingassayed when no contact is made with said compound of interest; whereby,enhanced reporter gene expression over said standard indicates that saidcompound of interest is an agonist of the IRAK-2 pathway, and diminishedreporter gene expression under said standard indicates that saidcompound of interest is an antagonist of the IRAK-2 pathway.
 18. Amethod for treating an individual in need of an increased level ofIRAK-2 activity comprising administering to said individual acomposition comprising an isolated polypeptide of claim
 11. 19. A methoduseful during diagnosis of a disorder, comprising: (a) measuring IRAK-2gene expression level in cells or body fluid of an individual; and (b)comparing the IRAK-2 gene expression level of said individual with astandard IRAK-2 gene expression level, whereby an increase or decreasein the IRAK-2 gene expression level over said standard is indicative ofan IRAK-2-related disorder.